The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) is an agricultural pest that ranges from the eastern Dakotas to Kansas and east to the Atlantic coast. The endosymbiotic bacteria Wolbachia has been detected in northern corn rootworm populations from east of the Mississippi River. Using the Wolbachia 16S rDNA, ftsZ and wsp genes a boundary was identified in central Illinois, between infected and uninfected populations with the infected populations found to the east of the boundary. Sequences of portions of the Wolbachia ftsZ and wsp genes have been obtained from four geographic locations of northern corn rootworm. Within infected northern corn rootworm populations, two strains have been detected. The 1,058-bp ftsZ sequences from northern corn rootworm indicate that both strains belong to the Wolbachia supergroup A. NCR Type 1 Wolbachia was found from eastern Illinois to Pennsylvania. NCR Type 2 Wolbachia occurs in central Illinois. The ≈600-bp wsp sequences from the two strains are also dramatically different. Strain differences in restriction fragment length polymorphism patterns of the Wolbachia-specific amplicons were used to determine the distribution of the strains. The boundary between these two strains of Wolbachia in native populations of northern corn rootworm correlates with a previously observed mitochondrial DNA genetic boundary in eastern Illinois, suggesting that the two Wolbachia strains are incompatible and little if any introgression occurs between the two infected populations. The results demonstrate that Wolbachia can influence the genetics of a major insect pest over a wide geographic area and that it may be driving reproductive isolation between populations of northern corn rootworm.