Interstate 65 begins its journey in Mobile at its junction with Interstate 10. From I-10, I-65 goes west of downtown Mobile and through its suburbs before turning northeast toward Montgomery. From the state's capital, I-65 heads north, bypassing Prattville and Clanton before going through Greater Birmingham. As the combined interstates of I-20/59 converge on Interstate 65 in Birmingham, many wrecks, and accidents that have occurred that include the melting of support beams of overpasses after 18-wheelers crashed and burned on two separate occasions, and the numerous collisions that happen every year, has led this interchange to be nicknamed, "Malfunction Junction".
From Birmingham, I-65 continues north toward Cullman and Decatur. At Decatur, motorists can take Interstate 565 eastward toward Huntsville.
From Decatur, the interstate continues northward through Athens to the Tennessee state line.
In 2004, following the death of President Ronald Reagan, a lengthy segment of I-65 from Jefferson County to Limestone County was designated "The Ronald Reagan Memorial Highway". The sign designating the north end of the segment includes a statement from Reagan's speech at Point Mallard Aquatic Center in nearby Decatur on July 4, 1984.
Hyundai's new automotive plant in Montgomery is located just off I-65. It can be accessed using the Pintlala-Hope Hull exit (number 164).
I-65 enters Tennessee from the south near the town of Ardmore, and passes through mostly rural territory for 65 miles. Its first city in Tennessee is Franklin. Then the route travels through Brentwood, Tennessee, downtown Nashville, Madison, Goodlettsville, White House, and exits near Portland into Kentucky to the North.
Interstate 65 enters the state five miles south of Franklin. It passes by the major cities of Bowling Green, Elizabethtown, and Louisville before exiting the state.
Throughout its length, it passes near Mammoth Cave National Park, Crystal Onyx Cave, Diamond Caverns, Bernheim Forest, the National Corvette Museum, and the Fort Knox Military Reservation.
It junctions with the four parkways. The first major junction is with the William H. Natcher Parkway at Bowling Green, followed by the Cumberland Parkway north of the city between Smiths Grove and Park City. At Elizabethtown, it has two more parkway interchanges with the Wendell H. Ford Western Kentucky Parkway and the Martha Layne Collins Bluegrass Parkway. Interstate 65 also has interchanges with I-265, I-264, I-64 and I-71.
The widest stretch of Interstate 65 in its entirety is in Louisville at the Kentucky Route 1065 (Outer Loop), where the mainline is 14 lanes wide. It crosses the Ohio River into Indiana on the John F. Kennedy Memorial Bridge.
At one time, the portion from Louisville to Elizabethtown was a toll road bearing the Kentucky Turnpike name. The bonds that financed the road have been retired, and tolls are no longer collected. All signs of the former turnpike have been removed.
On November 15, 2006, the stretch of I-65 from Bowling Green to Louisville was renamed the Abraham Lincoln Memorial Highway.
On February 12, 2007 a bill passed the Kentucky Senate to rename I-65 in Jefferson County the "Dr. Martin Luther King Jr. Expressway". Signage was posted July 25, 2007.
On July 15, 2007 Kentucky officially raised its speed limits on Interstate and State Parkway Highways to 70 miles per hour. Until that date, Kentucky was the only state along I-65's path that had a speed limit of 65 MPH.
Both of the Ford Motor Company truck plants in Louisville are accessible¡ªThe Explorer SUV plant is directly accessible from I-65 while the Kentucky Truck Plant, makers of the F-350 and Excursion SUV, is accessed via I-265.
In Bowling Green, I-65 comes within proximity of a GM plant, which makes the Chevrolet Corvette sports car, as well as the Cadillac XLR luxury roadster.
Interstate 65 enters the Hoosier state at Jeffersonville and Clarksville.
The section of Interstate 65 in downtown Indianapolis overlaps Interstate 70. The junctions are often referred to as the "North Split" and the "South Split", forming a section of interstate locally known as the "Inner Loop" or "Spaghetti Bowl" due to the visual complexity of the overlapping freeways.
In mid-March 2007, a six mile section of Interstate 70 from the North Split to Interstate 465 east of downtown was restricted to automobiles only for the "Super 70" project, a massive rebuild and expansion of that freeway. Trucks over 13 tons are forced to divert through Interstate 65 if coming from the north and use the circular Interstate 465 to the south to reconnect to Interstate 70 East. Westbound traffic from Interstate 70 must loop north or south along Interstate 465 to get to Interstates 65 or 70. This early part of the project is scheduled for completion in November 2007.
In the summer of 2003, the portion of Interstate 65 that runs concurrently with Interstate 70 was closed to all traffic due to the "HyperFix" project. During that time, a new asphalt surface was installed and the overpasses were upgraded.
Both the Super 70 and HyperFix projects were contracted to Walsh Construction of Chicago, Illinois.
In 1999, the 25 mile (40 km) segment of Interstate 65 between the two Interstate 465 interchanges was renamed the Kenneth "Babyface" Edmonds Highway.
Upon crossing into Lake County, IN over the Kanakkee River, the highway is know as the Casimir Pulaski Memorial Highway. It is known as this from that point to its northern terminus.
The northern terminus of Interstate 65 is only 1/8 mile (0.2 km) north of Interstate 90 (Indiana Toll Road). Prior to 2004, the interchange from Interstate 90 to Interstate 65 required making a physical left turn onto Interstate 65 via a traffic signal; traffic from Interstate 65 to Interstate 90 bypassed the traffic signal via an isolated right-turn lane. It has since been grade-separated.
In Lafayette, the Subaru of Indiana Automotive, Inc. plant is located at the junction of I-65 and SR-38. Supplier ArvinMeritor has several facilities near the highway in Columbus.