Yersinia enterocolitica (YE) is associated with several inflammatory gastrointestinal disorders. Pathogenic YE organisms are classified as biogroup 1B (high-virulence [HV] serovars) or biogroups 2 through 5 (low virulence [LV]). We developed the first molecular assay designed to distinguish between these groups and correlated the molecular results with histologic patterns of inflammation. Eleven known pathogenic YE culture isolates (6 biogroup 1B and 5 biogroups 2–5) and 6 YE-positive archival cases were subjected to polymerase chain reaction analysis using primer pairs targeting a strain-dependent variable region, allowing discrimination between biogroups with a single assay. All 11 known culture isolates were confirmed. Of the 6 archival cases, 4 were LV and 2 were HV. Histologic correlation revealed granulomatous inflammation in the LV cases and suppurative inflammation in the HV cases. This novel assay is useful for diagnosis using culture samples and archival tissues. It also could yield important information correlating YE epidemiology, pathogenesis, and morphology because these preliminary data suggest that LV strains may be associated with chronic granulomatous processes and HV strains with suppurative inflammation.