A simple method was developed to analyze the two morphologically indistinguishable host-associated strains of the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith). Total DNA extracted from the FAW corn and rice strains, as well as from the S. frugiperda cell line (SF9) was used to PCR amplify a 569 base pairs region of the mitochondrial gene cytochrome oxidase subunit I (COI). The amplified DNA from the Spodoptera corn strain and the SF9 cell line contained a restriction fragment length polymorphism (RFLP) marker, the MspI recognition site that was not present in the rice strain. This PCR-RFLP method does not require purification of mitochondrial DNA (mtDNA) or the use of radioactive isotopes, and differs from previous methods in that only a few nanograms of total DNA are needed to yield clear and accurate strain identification of individual insects.