Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a significant agricultural pest in the United States, affecting most notably sweet corn, Zea mays L., and turfgrass. Two strains exist that differ physiologically and behaviorally but are morphologically indistinguishable. Polymorphisms within the fall armyworm Triose phosphate isomerase gene (Tpi; EC 5.3.1.1) were characterized and used to develop a polymerase chain reaction-based method for discriminating fall armyworm strains. The Tpi markers were used in multilocus analysis to estimate interstrain hybrid frequency and their distribution in populations from the United States and Brazil. The results were compared with previous studies using different marker combinations to test hypotheses about interstrain mating behavior and to explain earlier observations of unusual strain distribution patterns. The importance of understanding the frequency and consequences of interstrain hybridization to deciphering the mechanism of strain divergence and the distribution of fall armyworm subpopulations in different habitats are discussed.