We studied the distribution of Pasteurella trehalosi genotypes isolated from oropharyngeal tissues of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in Waterton Lakes National Park, Alberta, Canada and Glacier National Park, Montana, USA. A separate radio-telemetry study indicated the bighorn metapopulation consisted of at least three neighborhoods of multiple ewe and ram social groups, with varying degrees of interchange among them. Genetic analysis using random amplified polymorphic DNA from 25 P. trehalosi isolates revealed three major genotypes. Our predictions were that genetic relatedness would be greatest among organisms collected in the same neighborhood, and that those collected from adjacent neighborhoods would show greater relatedness than those from distant or isolated neighborhoods. Results did not fully support these predictions. Nonetheless, the spatial distribution of P. trehalosi genotypes did vary with the observed neighborhood structure. Two of three genotypes occurred throughout the study area, but the third was found only in Waterton Lakes National Park. Because P. trehalosi is believed to be transmitted only via direct contact between individual sheep, this suggests the north Glacier neighborhood was further partitioned into two subpopulations. Overall, our results show the utility of using DNA from pathogens to elucidate the spatial structure of host populations.