This study investigated the genetic diversity among plum curculio, Conotrachelus nenuphar (Herbst), populations from 11 different geographic locations by using partial sequences of mitochondrial cytochrome oxidase I (mtCOI) gene. Polymerase chain reaction (PCR) products were 863 bp. In total, 23 unique haplotypes were identified from 50 tested samples. Haplotypes G (n = 5), L (n = 12), and T (n = 13) made up 60% of the 50 samples. Two distinct major clades were found in the phylogenetic analyses. Northern and southern clades were strongly supported (100/83 (MP/ML) and 100/62, respectively) by bootstraps. The mid-southern subclade within the southern clade was also moderately supported by bootstraps (73/57, MP/ML). The results of analyses of molecular variance (AMOVA) are consistent with phylogenetic analysis. The results suggested that the northern and the southern clades correspond with northern strain and southern strain of plum curculio. The three-group model of AMOVA test may suggest that there is a transitional group existing between northern and southern groups. In this study, the mtCOI sequence was highly informative as a molecular marker in that it could distinguish C. nenuphar from northern and southern geographic locations in the eastern United States.