We collected fish from the Missouri River, Montana, to investigate a 1984 finding of Mycobacterium chelonae (reclassified as M. salmoniphilum) from Mountain Whitefish (Prosopium williamsoni). Brown Trout (Salmo trutta), Mountain Whitefish, and Rainbow Trout (Oncorhynchus mykiss) were sampled from the Missouri River in 2009–2010, to examine for the presence of tissue nodules and granulomatous inflammation indicative of mycobacterial infection. Mycobacterium kansasii and a Mycobacterium sp. were detected by tissue smears and histology and identified by direct sequencing in tissue samples of 1.7% of Mountain Whitefish and 3.3% of Rainbow Trout. Genotypes were identified from 16S-23S rRNA. This study confirmed the continued presence of Mycobacterium infection in Mountain Whitefish from the Missouri River, with an updated diagnosis and identified infection in Rainbow Trout. This is the 1st documented case of Mycobacterium kansasii in wild fish and the freshwater documentation of a species similar to Mycobacterium sp. YM12, which was sequenced from a marine fish.