The soybean aphid (Aphis glycines Matsumura) is one of the most important insect pests of soybean [Glycine max (L.) Merr.] in North America, and three biotypes of the aphid have been confirmed. Genetic studies of the soybean aphid are needed to determine genetic diversity, movement pattern, biotype distribution and mapping of virulence genes for efficient control of the pest. Simple sequence repeats (SSR) markers are useful for population and classical genetic studies, but few are currently available for the soybean aphid. In this study, we designed primers for 342 genic-SSR markers from a dataset of more than 102 024 transcript reads generated by 454 GS FLX sequencing of a cDNA library of the soybean aphid. Two hundred forty-six markers generated PCR products of expected size and 26 were polymorphic among four pooled aphid DNA samples. An additional five markers that were fixed for two alleles among the pooled samples were found to be polymorphic when tested on 96 individual aphids. Sequencing of the PCR products generated by two polymorphic SSR markers revealed that the polymorphisms were strictly because of variations in the SSR repeats among the aphids tested. The genetic diversity among 96 soybean aphids, 24 each from two field collections (South Dakota and Michigan, biotype unknown) and two laboratory colonies [biotype 1 (B1) and biotype 2 (B2)], was assessed with 29 polymorphic SSR markers. These markers discriminated laboratory colonies from field collections and field collections from different states. The genic-SSR markers developed and validated in this study will be a significant addition to the limited number of SSR markers, mostly genomic-SSR, currently available for the soybean aphid. These markers will be useful for genetic studies, including population genetics, genetic and QTL mapping, migration and biotype differentiation of the soybean aphid.