Populations on the periphery of a species’ range tend to contain lower genetic variation and increased genetic differentiation compared to populations at the core of a species range, although some exceptions to this generalization occur. The blister beetle Gnathium minimum (Say) exhibits a wide-ranging distribution in the western United States but has peripheral or disjunct populations in Mexico, Florida, and Wisconsin. We used amplified fragment length polymorphism (AFLP) to compare the genetic variation and magnitude of genetic differentiation of the Wisconsin peripheral population to western core populations (Colorado, Kansas, New Mexico, and Texas). The proportion of polymorphic loci was 53.6 and 54.3, and expected heterozygosity 0.1864 and 0.1933 for the Kansas/Colorado (n = 87) and New Mexico/Texas (n = 35) regions, respectively. Specimens from Wisconsin (n = 121) had a lower proportion of polymorphic loci (38.4) and expected heterozygosity (0.1475). Genetic cluster estimation with GENELAND and F ST values showed greater genetic differentiation among the sampling locations within Wisconsin compared to core regions. Significant isolation-by-distance (IBD) was also observed in Wisconsin but not within the core regions. Lower genetic variation and increased isolation may reduce the Wisconsin population’s ability to respond to change, thereby increasing their susceptibility to extinction.