The ecological consequences of impoundment construction on riparian systems throughout the U.S. Southwest has profoundly affected a variety of organisms, including many amphibians. To better understand the current extent of hybridization and changes in genetic composition over time in Bufo (Anaxyrus) woodhousii and Bufo microscaphus, we used microsatellite loci to evaluate 260 individuals representing 10 total populations constituting B. woodhousii, B. microscaphus, and putative hybrids along the Agua Fria River in Arizona during two time periods (1992–97 and 2009–10). Consistent with prior work with these two anurans documenting unidirectional replacement or genetic introgression, we predicted that microsatellites would provide evidence of directional introgression of B. woodhousii into B. microscaphus. The putative hybrid populations exhibited the highest number of alleles, and B. microscaphus exhibited the lowest number of alleles. Structure analysis indicated K = 2 as the best-fit population number for both time periods. All pairwise F-statistics were highly significant, corroborating differentiation among populations as inferred by structure analysis. The principal coordinates analysis demonstrated three distinct clusters of individuals that corresponded strongly with prior morphological and mitochondrial assignments within this region over both sampling periods. Our findings indicate that the genetic identity of B. microscaphus remains distinct from B. woodhousii and the hybrids, suggesting that the genetic structure of the corresponding populations has remained intact. Bufo woodhousii has not replaced B. microscaphus along the Agua Fria River beyond those habitats directly associated with impoundment construction.