Fall armyworm is a significant agricultural pest in the Western Hemisphere and an important system for studying Lepidopteran migration and speciation. Critical to these investigations are methods that can differentiate between two morphologically indistinguishable strains that differ in their choice of plant host. In a previous study, haplotypes of the fall armyworm Triose phosphate isomerase gene (Tpi; EC 5.3.1.1) were shown to be effective indicators of strain identity. However, the method had technical complications that made it expensive to apply on the scale needed for most population studies. The focus of this paper was to develop a more efficient and cost-effective procedure. By combining polymerase chain reaction (PCR) amplification and restriction enzyme digestion, a strain-specific polymorphism in the fall armyworm Tpi locus can be characterized from single specimens without the need of DNA sequencing. This method was more accurate under some circumstances than COI haplotyping, the current method of choice for population studies. The modified Tpi method was used to expand upon previous indications that interstrain hybridization occurs asymmetrically, to confirm observations of seasonal periodicity in the proportions of the two strains infesting Florida cornfields, and to demonstrate that certain instances where strain specificity to their host plants appeared to be compromised were because of variability in the association of the COI markers to strain identity. The results indicate that the application of both Tpi and COI analyses to the study of fall armyworm populations is now practical and should facilitate the mapping and study of fall armyworm subpopulations in the United States.