Ecological disturbance has often been used to explain the breakdown of reproductive isolation in closely related taxa, especially in North American toads (family Bufonidae). In the Southwest, impoundment construction has altered many riparian systems, and apparently resulted in recent sympatry and hybridization of Bufo (Anaxyrus) microscaphus (Arizona toad) and B. woodhousii (Woodhouse's toad). Our specific objective was to generate microsatellite data at eight highly polymorphic nuclear loci for populations of B. microscaphus, B. woodhousii and putative hybrids to evaluate the hybrid zone between these two anurans along the Agua Fria River near Phoenix, Arizona. The microsatellite loci characterized here were modified from microsatellites originally developed for the endangered arroyo toad, B. californicus. These eight highly polymorphic loci were tested on populations of B. microscaphus, B. woodhousii, and putative hybrids in the watershed of the Agua Fria River in Arizona, USA. All loci were highly variable among the 66 samples evaluated. Structure analysis indicated that K=2, suggesting that B. woodhousii and B. microscaphus were distinct with hybrid individuals present, and the F-statistics corroborate these findings. Finally, PCoA analysis separated the three groups of individuals into distinct clusters with little overlap between species but some overlap of hybrids with B. microcaphus. Our findings suggest that these nuclear markers are useful for future population genetic investigations in this group.