Technical Abstract: The northern corn rootworm (Diabrotica barberi) in eastern and central North America exhibits at least three distinct populations with respect to Wolbachia infection: uninfected; singly-infected; multi-infected. The infected states are associated with different mtDNA haplotypes and reduced mt variability. Both wsp and MLST sequencing indicated that the multiple infections include at least five distinct strains. The recovery of the different wsp hypervariable regions among 55 clones obtained with universal primers from several individuals was unequal. The most common was obtained 33 times, the least common only once. Possible explanations are that the frequency of each strain is not the same within each insect. An alternative possibility is that not all individuals are infected with all five strains and that different animals contain different combinations of the strains. Under this scenario the results might suggest that some strains are rare in the population. The two explanations are not mutually exclusive. We designed a group of primers specific for the different HV regions. The plan was to use various primer combinations that could amplify each of the five strains to determine their presence or absence in single beetles. The primers were tested on beetles that had previously shown to be harboring certain wsp variants. Some of these amplicons were sequenced to verify that they contained the anticipated HV regions. The approach is being used to measure the distribution of the wsp strains.