MNA operates approximately 527 miles (848 km) of line in Arkansas, Kansas, and Missouri. Its main line extends 384.1 miles (plus 40 miles (64 km) of trackage rights on Union Pacific Railroad lines) from Kansas City, Missouri, to Newport in Jackson County in northeastern Arkansas. (MNA interchanges with Union Pacific at both points). MNA also interchanges with BNSF Railway at Carthage, Aurora, Springfield, and Joplin, Missouri, and with the Kansas City Southern Railway at Joplin. Although the line still interchanges with the BNSF mainline at Lamar, the line no longer has a connection to the Pittsburg, KS to Nevada, MO. line, as the majority of that line, currently owned by the South Kansas and Oklahoma Railroad, is abandoned on the Missouri side of the state line.
MNA also operates several miles of industrial trackage in the city of Springfield; this segment is disconnected from the rest of the MNA system and traffic is hauled via BNSF to and from the Aurora interchange.
The Branson Scenic Railway operates the "Ozark Zephyr" from Branson, Missouri. Trains operate mostly south into Arkansas but occasionally north to Galena, Missouri, depending on MNA traffic. The restored 1906 depot is across from Branson Landing in historic downtown Branson.
The White River Scenic Railroad formerly operated an excursion train from Flippin to Calico Rock, Arkansas.