Pseudoroegneria spicata (Poaceae: Triticeae) is an abundant, allogamous species widely adapted to the temperate, semiarid steppe and open woodland regions of western North America. Amplified fragment length polymorphism (AFLP), model-based Bayesian clustering, and other methods of hypothesis testing were used to investigate genetic diversity and population structure among 565 P. spicata plants from 82 localities representing much of the species distribution. Comparisons with four Asiatic Pseudoroegneria species and two North American Elymus wawawaiensis accessions demonstrate cohesiveness in P. spicata. However, P. spicata genotypes group by locality and geographic region based on genetic distance analysis. Average DNA polymorphism among P. spicata localities was significantly correlated (r = 0.58) with geographical distance. The optimum Bayesian cluster model included 21 P. spicata groups, indicating that dispersal among sampling locations was not sufficient to group genotypes into one unstructured population. Approximately 18.3% of the DNA polymorphism was partitioned among the 21 regional groups, 14.9% among localities within groups, and 66.8% within accessions. Average DNA polymorphism among Bayesian groups was correlated (r = 0.53) with the average geographic distance among Bayesian groups, which partly reflects isolation by distance. However, conspicuous regional boundaries were discernable among several divergent genetic groups.