Wolbachia are a rickettsial type bacteria that have been implicated as a cause of reproductive disruption and alteration in many insect species. Polymerase chain reaction assays of the 16S rRNA gene were conducted to reveal the prevalence of Wolbachia in 14 Diabroticite species, 12 Diabrotica, and two Acalymma. Assays revealed the presence of Wolbachia infection in three Diabrotica species; D. virgifera virgifera LeConte (all 40 individuals), D. cristata (Harris) (three of 25 individuals), D. lemniscata LeConte (all 20 individuals), and the two Acalymma species, A. blandulum (LeConte) (all 20 individuals), A. vittatum (F.) (one of 20 individuals). The identity of the Wolbachia strain was determined by sequencing the 16S rRNA and ftsZ genes as well as a restriction enzyme digest of the ftsZ gene. Distances and neighbor-joining trees on the Kimura 2-parameter measure as well as BlastN searches in GenBank revealed that the strain of Wolbachia infecting D. lemniscata, D. v. virgifera, A. blandulum, and A. vittatum are most likely the same strain of Wolbachia within the division A group. The strain of Wolbachia infecting D. cristata also clusters within the A group, but it is different from that found in the other four Diabroticites. Implications of these infections are also discussed.