We studied the distribution patterns of Wolbachia infection associated with plum curculio strains in eight states of the eastern United States. The presence of the Wolbachia-specific gene wsp identified infections of this endosymbiont in 97.8% of the 93 samples tested. Three distinct Wolbachia strains were identified. The strains wCne1 (593 bp) and wCne2 (593 bp) were 97% identical, and their sequences were both 84% identical with wCne3 (590 bp). BLASTN searches through GenBank showed strong similarities between the wsp sequences of the three strains compared with Wolbachia sequenced from other hosts. Degree of similarity with sequences in other Wolbachia strains is discussed. Polymerase chain reaction-restriction fragment length polymorphism was used for superinfection detection. Of 93 samples, 15 (16.1%), 21 (22.6%), 19 (20.4%), and 36 (38.7%) samples were infected by wCne1, wCne2, wCne1 2, and wCne3, respectively. Only two (2.2%) samples had no infection. The wCne3 strain was always present as a single infection. Wolbachia strains approximate the distribution of plum curculio strains: northern strain infected with wCne1 and wCne2 strains in supergroup B, and southern strain infected with wCne3 strain in supergroup A, with the mid-Atlantic region as the convergence area. Based on haplotype distribution of plum curculio mitochondrial cytochrome oxidase I, there was a closer relation of the mid-southern plum curculio clade to the far-southern clade than to the northern clade. However, Wolbachia symbionts in mid-southern plum curculio are more closely related to those in northern plum curculio than to those in far-southern plum curculio. The relationship of Wolbachia infection with reproductive incompatibility between plum curculio populations was also discussed.