This is the first report of sequence-specific microsatellite markers (simple sequence repeats [SSRs]) of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), an economically important pest of crops on the Americas. We isolated 192 microsatellite markers by using pyrosequencing and screened 15 individuals from eight isofamilies collected from three geographical areas: Puerto Rico (PR), Texas (TX), and Mississippi (MS). Isofamilies resistant to Cry toxins from Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae) also were included. Cluster analysis was performed to determine the potential use of these SSRs in discriminating populations, and colonies were grouped with a reliability of 100% estimated by bootstrap. In this analysis, colonies from TX grouped away from those from PR, but the two MS isofamilies grouped with TX and PR separately. Genetic distance within isofamilies ranged between 0.22 and 0.56, and the minimum distance between isofamilies was 0.83. Unique pattern informative combination (UPIC) scores were calculated, and the 80 SSR markers that had UPIC scores of ≥1 are listed according to their discriminating potential. UPIC scores allow reducing costs by choosing fewer and highly informative markers for future studies. From the best 125 markers, 103 had a maximum of two alleles per sample, making them ideal candidates for population genetic studies. BLAST screening of the sequences points to potential biological meaning of marker polymorphisms. The percentage of alleles shared by the three geographic areas was 14%. The markers reported will significantly enrich the pool of molecular markers available for S. frugiperda. In addition, they could be used for monitoring migration of populations, in the development of biocontrol agents and for management practices in general.