Mycoplasma hyopneumoniae isolated from different pig herds showed variation in disease potential. This is not unexpected since differences in disease symptoms occur widely in the field. There have been few studies of this type, however, because of the difficulty in isolating the organism from clinical samples and growing it in the laboratory. This is also one of the few studies where pure cultures have been used to establish significant disease in clean (pathogen-free) pigs. During the course of these studies, we also demonstrated changes in the genome (chromosome) of the organism in an unexpected region used for diagnostics. This was evident when a newly developed real time polymerase chain reaction test (RT-PCR) was used to study the field strains examined in this study. The advantage of RT-PCR is that quantitative data can be obtained in a short period of time in comparison to the CCU test that requires weeks to complete. This particular RT-PCR assay targets a small segment of the region that has been used previously as a PCR target. That small DNA sequence was found to be in a region that differs from the challenge strain 232, preventing detection by the RT-PCR assay. The nested PCR, however continued to detect all field strains further confirming its utility as a diagnostic tool. These studies demonstrate the need to further test all PCR-based diagnostics with more field strains to ensure accuracy and reproducibility. Variation in a second genome region was both expected and confirmed. This region of the genome has shown variation in the past, but it was clear that a combination of the two tests could enable differentiation of field strains. The sensitivity of PCR diagnostics may even allow differentiation in lieu of culture, a significant advance. Finally, we were able to establish the conditions needed to perform genome-wide comparisons of M. hyopneumoniae field strains using microarrays. Although these tests were not completed, our laboratory is well positioned to perform these studies in the near future. These will be the first such studies with an important pig pathogen and promises to enhance our understanding of the mechanisms involved in porcine enzootic pneumonia.