The lesser peach tree borer, Synanthedon pictipes (LPTB), belongs to the economically important Lepidopteran family Sesiidae. No studies on genetic variation or population structure on the genus Snyanthedon have been previously published. We examined DNA sequence variation in a 603 bp region of the mitochondrial cytochrome oxidase I gene (COI), tRNA-leu and cytochrome oxidase II gene (COII) from three LPTB populations in Arkansas. From 114 LPTB collected from three populations, a total of 53 nucleotide positions were polymorphic, and 12 distinct haplotypes were observed. The most frequent haplotype occurred in 88% of the sampled LPTB's and in all three populations. Sequence divergence among haplotypes ranged from 0.2% to 8.8%. According to the standard molecular clock proposed for lepidopteran mtDNA, the haplotypes have been diverging for up to 2.5 million years. The greatest amount of haplotype diversity was observed in the Fayetteville population where borer management is not maintained. High levels of gene flow were observed among the Clarksville, Springdale and Fayetteville populations suggesting the LPTB has a broad dispersal range. Examination of the genealogical relationships and phylogenetic analysis of the 12 haplotypes supports the existence of three genetically distinct but morphologically indistinguishable subspecies.