Larvae of many species of Sesiidae, the clearwing moths, are important pests in commercial nurseries, urban landscapes, timber stands, vineyards, and orchards. They cause economic loss by larval boring in stems and roots of herbaceous and woody plants. Researchers and growers often monitor for the presence of economically important sesiid adults with pheromone traps. These traps often attract more than one species of Sesiidae and specimens often degrade making identification difficult or impossible. This can cause problems in monitoring programs where species-specific control programs are used. Polymerase chain reaction (PCR) was used to amplify a 606-bp region of the mitochondrial DNA cytochrome oxidase I (COI), tRNA leucine, and COII gene. This region exhibited 7.7-19.5% genetic variability among 8 species of Sesiidae. Samples were sequenced and restriction sites identified. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis was conducted on 8 species of Sesiidae, Melittia satyriniformis, Paranthrene simulans, Pennisetia marginata, Synanthedon pictipes, S. exitiosa, S. scitula, S. rileyana, and Vitacea polistiformis, with 2 restriction enzymes, Dra I and Hinf I. This method is time efficient requiring less than 8 h to perform and cost efficient with each sample about $1. PCR-RFLP provides an accurate method to differentiate 8 species of adult clearwing moths commonly found in traps baited with commercially available pheromone lures.