Abstract: A Mycoplasma bovis PCR-based genotyping method was developed that exploits the proximity of insertion sequences (IS) within the genome by using outward facing primers that selectively amplify sequences between IS elements. The method was applied to 16 field isolates of M. bovis, originating from pneumonic lung or arthritic joints, collected from the United States (Iowa or Kansas) between 2004 and 2005. The genomic fingerprints generated 14 distinct amplification profiles consisting of 4-8 fragments ranging in size from 200-3000 bp. Three isolates presented identical patterns and were isolated from two calves (one calf with pneumonic lung and the other with both pneumonic lung and arthritic joint) from a single farm during an outbreak and probably represent multiple infections with the same genotype. To demonstrate the stability of IS markers for molecular fingerprinting, 3 of the 16 field isolates were subjected to high number passage which resulted in patterns identical to the initial isolates.