Three restriction site markers from the mitochondrial genome are described for the toads Bufo microscaphus and B. woodhousii. Markers were resolved from DNA sequence data representing segments of cytochrome b (cytb) and 16S ribosomal genes. Two diagnostic restriction sites (BsmA I; SfaN I) characterize B. microscaphus for cytb, whereas a third restriction site (Tsp509 I) distinguishes B. woodhousii for 16S. Restriction digests of PCR-amplified cytb and 16S segments provide an economical, nondestructive assay with which to identify mtDNA haplotypes for these toads and their hybrids. The mtDNA markers complement diagnostic allozyme and call characters, offering an additional approach to examine hybridization between the two species in central Arizona.