The company operates over 580 miles of track. The railroad's mainline is roughly a straight line between Council Bluffs, Iowa, and Chicago, Illinois, with a branch line connecting Bureau to Peoria, Illinois. The mainline is separated into 4 Subdivisions:
Council Bluffs Subdivision (Council Bluffs to Des Moines, Iowa)
Newton Subdivision (East Des Moines to South Amana, Iowa)
Iowa City Subdivision (South Amana to Silvis, Illinois)
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Blue Island Subdivision (Silvis to Bureau Junction, Illinois)
The railroad also includes the:
Peoria Subdivision (Bureau Junction to Peoria, Illinois)
Cedar Rapids Subdivision (South Amana to Cedar Rapids, Iowa). This line is owned by the Cedar Rapids and Iowa City Railroad, but dispatched and operated by the Iowa Interstate.
The Iowa Interstate operates from Bureau to Chicago over the former Rock Island tracks controlled by CSX and Metra. In Chicago the IAIS owns two yard facilities, Burr Oak Yard and Evans Yard (the site of former rail car builder Evans), both in Blue Island, Illinois.
Trains are dispatched from the company's HQ in Cedar Rapids, Iowa, where a new dispatching office was completed in 2016. IAIS uses Wabtec's Train Management and Dispatching System (TMDS), the same dispatching software used by several Class I railroads, including the BNSF and KCS. When the IAIS took control of the track, the former Rock Island signal system was already damaged beyond repair due to sitting dormant for several years. Operations on the railroad are primarily controlled by track warrants rather than signals as a result.
The Iowa Interstate is unique in that it is the only Class II railroad in the US that has connections to every Class I railroad, affording its customers a reach not offered by other regional railroads.
The railroad also maintains two intermodal operations at either end of its line in Chicago and Council Bluffs. The operation in Council Bluffs is also used by the Union Pacific, with IAIS crews interchanging with UP several times a day.
IAIS subsidiary Rail Traffic Control formerly provided consulting services for dispatching and operating small- to medium-sized railroads worldwide.
In 2004, IAIS was awarded the E. H. Harriman Award for its safe operational record.