The northern corn rootworm (Diabrotica barberi Smith & Lawrence) in eastern and central North America exhibits at least three distinct populations with respect to Wolbachia infection: uninfected; singly infected; multiply infected. The infected states are associated with different mtDNA haplotypes and reduced mtDNA variability. Multilocus sequence typing (MLST) was used to define strain types and examine the diversity of Wolbachia infecting northern corn rootworm. Four of the five MLST genes (coxA, hcpA, fbpA, and ftsZ) were represented by three alleles each. The remaining MLST gene (gatB) had four to six alleles. The uncertainty arose from whether to call two small clusters new alleles or artifacts of the amplification process. Singly infected individuals had a common set of alleles that defined one strain (wBarB). This strain was also a component of the multiple infections. A second strain (wBarA), restricted to the multiple infections, was defined by alleles that appeared with substantially greater frequency for each of the five loci. By default a third strain would comprise the less frequent third allele of the four loci, but it is not possible to determine to which gatB allele they are linked. Therefore, the strain is not fully defined. The diversity of gatB indicates that four to six Wolbachia strains are present in northern corn rootworm, a number that is similar to the five variants reported previously for the wsp gene. These results also highlight the very real difficulty of using the MLST system to define specific strains in a multiply infected host.