Restriction-endonuclease analyses of mitochondrial DNAs from all six color-pattern classes (A-F) of the parthenogenetic lizard Cnemidophorus tesselatus yield estimates of nucleotide divergence that are extremely low (π = 0.06%). In digests of 75 C. tesselatus mtDNAs with 20 different restriction enzymes, only four cleavage-site differences were noted, three of which were found only in pattern class F. The near-identity of these mitochondrial DNAs with those from C. tigris marmoratus shows unequivocally that C. t. marmoratus was the species to which the maternal parent(s) of all C. tesselatus belonged. Mitochondrial-DNA analyses of another unisexual species, C. neomexicanus, led to the same conclusion. Mitochondrial DNAs from 96 individuals of these three species were extensively analyzed for cleavage-site differences; only 13 were found. The low interspecific sequence diversity found within C. neomexicanus and the C. tesselatus complex suggests a recent origin for both. Based on diversity data for mitochondrial DNA and allozymes, we estimate that a minimum of two hybridizations were required to produce all diploid C. tesselatus (C-F), followed by at least two more to generate the triploids (A and B). These data and those presented in the two accompanying papers indicate that events leading to parthenogenesis in Cnemidophorus are rare and strengthen the hypothesis that interspecific hybridization is a necessary, causal event in its establishment.