A disease outbreak of high morbidity and high mortality in bison ( Bison bison) was investigated. Clinical signs included lameness, swollen joints, respiratory distress, and lethargy. Fifty-three of 194 animals died. Cows between 5 and 10 years of age were the most affected group, in which 40 of 88 animals died. Necropsies were performed on several animals. There were abscesses in the lung and liver, as well as fibrinosuppurative pleuritis, polyarthritis, and disseminated microabscesses in various organs. No significant bacteria were isolated by routine aerobic cultures of lung and liver from 2 representative cases. However, Mycoplasma cultures were positive. Polymerase chain reaction tests on the isolated bacteria were positive for Mycoplasma bovis. Histologically, the abscesses were characterized by areas of necrosis with variable mineralization rimmed by granulomatous inflammation and fibrous tissue. No new animals had been introduced into the herd, but a cattle herd was present adjacent to the affected bison herd. Two restriction fragment length polymorphism techniques were used to compare the bison isolate and another bison isolate from an outbreak in North Dakota with a field isolate of M. bovis from cattle and with a laboratory control strain of M. bovis; the isolates and control strain were found to be similar. The isolates and the control were sequenced and compared with sequences in GenBank. Bison isolates were more than 99% homologous to M. bovis sequences in GenBank. It was concluded that M. bovis in bison can cause disseminated infection with a high morbidity and mortality and that bison isolates are similar to bovine M. bovis isolates.