Smallmouth Bass (Micropterus dolomieu) and Largemouth Bass (M. salmoides) are congeneric species that co-occur in many lentic and lotic systems, but have never been documented to hybridize in the wild. In 2003, a long-term study of reproductive ecology of M. dolomieu encountered a nest defended by a male M. dolomieu with two distinctly different size sets of eggs. The size range of one set of eggs was consistent with that observed for M. dolomieu, but the size range of the second set of eggs was too small for M. dolomieu and was within the size range for M. salmoides. A sample of fin tissue from the male guarding the brood and a subsample of both size classes of offspring were collected. Molecular genetic techniques (protein electrophoresis, microsatellite analysis, and RFLP analysis of mtDNA) confirmed that progeny from the larger eggs were from a mating between the nest-guarding male and a female M. dolomieu and that those from the smaller eggs were from a mating between the nest-guarding male and a female M. salmoides. The most plausible explanation for the breakdown of prezygotic isolating mechanisms is that the very low abundance of adult M. salmoides precluded conspecific mating for the female M. salmoides. (c) 2010 by the American Society of Ichthyologists and Herpetologists.