Historically, many population genetics studies have utilized microsatellite markers sampled at random from the genome and presumed to be selectively neutral. Recent studies, however, have shown that microsatellites can occur in transcribed regions, where they are more likely to be under selection. In this study, we mined microsatellites from transcriptomes generated by 454-pyrosequencing for three vertebrate species: lake sturgeon (Acipenser fulvescens), tiger salamander (Ambystoma tigrinum), and kangaroo rat (Dipodomys spectabilis). We evaluated (i) the occurrence of microsatellites across species; (ii) whether particular gene ontology terms were over-represented in genes that contained microsatellites; (iii) whether repeat motifs were located in untranslated regions or coding sequences of genes; and (iv) in silico polymorphism. Microsatellites were less common in tiger salamanders than in either lake sturgeon or kangaroo rats. Across libraries, trinucleotides were found more frequently than any other motif type, presumably because they do not cause frameshift mutations. By evaluating variation across reads assembled to a given contig, we were able to identify repeat motifs likely to be polymorphic. Our study represents one of the first comparative data sets on the distribution of vertebrate microsatellites within expressed genes. Our results reinforce the idea that microsatellites do not always occur in noncoding DNA, but commonly occur in expressed genes.