[2] Indiana Southern Railroad operates 186 miles (299 km) of track[2] between Indianapolis and Evansville.

From Mars Hill (a neighborhood on the southwest side of Indianapolis) southwest through Martinsville and Spencer to Bee Hunter in Greene County, the ISRR runs on tracks that once made up the majority of the former Indianapolis & Vincennes Branch of the Pennsylvania Railroad.

The Indiana Southern began operations with a fleet of 10 ex-CSX EMD GP40 locomotives which were rebuilt without dynamic brakes and identified as GP40-1s.

As of 2018, the ISRR operated 10 ex-BNSF EMD SD40-2 locomotives acquired from First Union Railway Equipment in 2013 after the railroad became Genesee & Wyoming property.

A siding between Whitaker and Gosport is regularly used for car storage; as is a small yard in Worthington on the former New York Central trackage.