The fall armyworm, Spodoptera frugiperda, consists of two host strains (rice and corn) that differ in developmental, physiological and behavioural characteristics. However, because the strains are morphologically indistinguishable the investigation of strain-specific behaviour, particularly in the wild, is very difficult. This has spurred the isolation of diagnostic molecular markers. FR sequences are tandem-repeat genetic elements found in large clusters only in the rice strain. To facilitate their use as a strain marker we genetically mapped FR clusters and found they localized to the sex chromosomes. This represents one of the first examples of chromosome mapping in fall armyworm. The FR sequence and a strain-specific mitochondrial marker were then used to examine the distribution of different marker combinations in field specimens. These studies identified significant barriers to interstrain mating in the wild, specifically that corn strain females rarely, if ever, mate with rice strain males. The data also suggest that only a genetically distinct subset of the overwintering rice strain population in Florida annually migrate to Georgia. These studies demonstrate that the availability of genetically characterized molecular markers for strain identity makes possible studies on fall armyworm biology in the wild previously considered unfeasible.