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.