To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees.