Wolbachia are alpha protobacteria that often cause alteration of host reproductive biology. This behavior has led to the hypothesis that Wolbachia contribute to reproductive isolation within species. Diabrotica virgifera zeae, and D. virgifera virgifera are currently classified as subspecies; both are key pests of maize. D. v. zeae is distributed from Central America to Oklahoma in the United States. D. v. virgifera is found from northern Mexico to southeastern Canada. This study was conducted to 1) infer the pattern of Wolbachia infection in D. virgifera (sensu lato) in Central and North America (amplified 16S rRNA and ftsZ genes) and, 2) to contrast intraspecific variation of Wolbachia infection with phylogeographic variation in the mtDNA COI gene to reconstruct the history of differentiation and expansion of living populations of the species. Wolbachia was found in D.v.zeae in Mexico. The strain belongs to Wolbachia division A, but is different from strains present in United States populations of D. v. virgifera. Nested Clade Phylogeographic Analysis and phylogenetic analysis of mtDNA sequence data collectively supported the geographic separation of D. virgifera into two phylogroups along a North-South axis. The geographic distribution of Wolbachia in D. virgifera was poorly correlated with D. virgifera COI mtDNA subdivision patterns over the geographic area included in this study. Results are discussed within the context of current hypotheses pertaining to the interaction of Wolbachia and D. virgifera subspecies.