The purpose of these studies was to investigate the implications of variability in virulence, genetics and antigenic profiles among field isolates of Mycoplasma hyopneumoniae. Increased virulence was compared with in vitro and in vivo growth characteristics of the organism and with immune parameters in the pig, although none was found to be significantly correlated. The impact of proteomic and genetic variation among field isolates on diagnostic assays was examined by both in vivo and in vitro studies. Three enzyme-linked immunosorbent assays (ELISAs) used in the United States were compared and shown to not detect all isolates of M. hyopneumoniae equally. Similarly, polymerase chain reaction (PCR) assays were tested against a panel of M. hyopneumoniae field isolates and it was shown that some isolates are not detected by all published PCR assays. Therefore, two new real-time PCR assays were developed that appear to be both sensitive and specific for M. hyopneumoniae. The ability of a commercial vaccine to protect against field isolates of M. hyopneumoniae was evaluated in a challenge study using two recent field isolates. One isolate produced nearly twice as many macroscopic lung lesions as the other isolate, but vaccinated pigs had less pneumonia compared to non-vaccinated pigs in both challenge groups. Together these results demonstrate that differences exist among isolates of M. hyopneumoniae in the field at the genetic and antigenic level and that these differences impact the virulence of the organism, diagnostic capabilities in the pig, and the prevention of M. hyopneumoniae-associated disease.