Mycoplasma bovis (M. bovis) is an important bacterial pathogen of cattle that commonly causes pneumonia, mastitis, and arthritis. M. bovis has recently emerged as a primary pathogen in American bison, causing significant morbidity and mortality. We explore mycoplasmas as potential threats for emerging infectious diseases and large-scale outbreaks in wildlife. We review M. bovis in cattle, how M. bovis infections manifest in bison, current practices and knowledge for management in bison, and identify gaps in testing protocols. Evidence suggests that M. bovis can be carried asymptomatically in bison. Asymptomatic infections could be important sources of outbreaks; detection of such infections is thus critical to disease management. Little is known about the optimal sampling technique for ante-mortem detection of M. bovis in bison. Nasopharyngeal swabbing is recommended in cattle; support for a similar technique has not been established in bison. We compared paired superficial nasal swabs and nasopharyngeal swabs in sick and apparently healthy bison, then used PCR to detect M. bovis in 86 out of 957 bison (8.9%) from 16 herds. Of bison testing positive on at least one swab type, we detected M. bovis in 82.5% of deep nasopharyngeal swabs (n=71) and 41.8% of superficial nasal swabs (n=36). Agreement between swabs for positive bison was 24.4% (n=21). We conclude that nasopharyngeal swabbing is most sensitive for detection of M. bovis in bison. Determining optimal sampling technique and understanding detection limitations can improve M. bovis surveillance in bison, impacting the economics and conservation of bison across North America.