The genetic structures of the Iowa Corn Borer Synthetic #1 (CB) and Iowa Stiff Stalk Synthetic (SS) maize (Zea mays L.) populations are important because these populations serve as the model for development of modern commercial hybrids. In 1949, CB and SS were used to start a reciprocal recurrent selection (RRS) breeding program at Iowa State University. This study was conducted to analyze more thoroughly the genetic diversity within this RRS program and illustrate how the RRS program has changed over time at the molecular level. The progress of this program was measured by analyzing the variation at 86 SSR loci among 28 progenitor lines and 30 plants sampled from each of seven cycles (Cycle 0, Cycle 1, Cycle 3, Cycle 6, Cycle 9, Cycle 12, and Cycle 15) in each population. The progenitors of these populations show a high amount of variation on the basis of expected heterozygosity (0.557). As the RRS program proceeded, this variation decreased (Cycle 15, 0.245). In total, a larger amount of genetic variation was found among plants within cycles (66%) than among cycles (13%) or between populations (21%). The repartitioning of variation from within populations (96% in progenitors) to between populations (58% in Cycle 15) over time is consistent with theoretical expectations of divergence between the populations. By sampling intermediate time points, we gained a comprehensive genetic view of CB and SS permitting evaluation of the molecular-level changes occurring as a result of reciprocal recurrent selection.