Northern corn rootworm (Diabrotica barberi)(NCR) populations in the USA are infected with at least 3 strains of the endosymbiont, Wolbachia. NCR from central Illinois appears to be singly infected with a strain wBar2. NCR from eastern Illinois to Pennsylvania appear to harbor at least 2 different strains designated wBar1 and wBar3. NCR from western Illinois through the Great Plains are uninfected. A previously discovered mtDNA demarcation zone in USA populations of appears to be associated with the different Wolbachia strains. The mtDNA haplotype boundary in eastern Illinois correlates with boundary of singly infected wBar2 individuals and the doubly infected beetles. Sequences of a portion of the ftsZ and wsp genes revealed that wBar1 is nearly identical (<0.3% divergence) to the Wolbachia strain found in the western corn rootworm (D virgifera virgifera). WBar1 versus wBar2 exhibit the greatest nucleotide sequence divergence at 3.1% (ftsZ) and >10% (wsp). WBar3 is intermediate between the other two strains. Compared to uninfected populations, NCR mtDNA diversity is reduced in regions with substantial Wolbachia infection indicating that Wolbachia sweeps have occurred. Where the mtDNA clades adjoin some individuals are infected with all three Wolbachia strains. It is not known if these individuals are fertile. Work is underway to determine if NCR harbors any additional Wolbachia.