The occurrence of rare or novel alleles has been documented in at least 23 different hybrid zones spanning vertebrate and invertebrate taxa. As most novel alleles either occur at high frequencies in hybrid populations or are exclusively restricted to hybrids, it has seemed probable that hybridization has a role in their origin; however, the molecular nature of these novel alleles and the mechanisms responsible for their origin remain obscure. We examined the complete coding sequences of six alleles of alcohol dehydrogenase in a mammalian hybrid zone between two species of pocket gophers (Geomys). One of these sequences encodes a novel electromorph that had been identified in earlier allozyme studies; this novel allele differs from one of the parental alleles by a single base substitution. This substitution generates an amino acid replacement that affects the net charge of the translated protein. This resultant charge change is congruent with the observed allozyme mobility patterns. Our data thus provide evidence for simple DNA substitution as a mechanism for the origin of this novel hybrid-zone allele.