Stream flow modifications, such as reduced spring flow and construction of low-head dams, have led to declines in the headwater catfish (Ictalurus lupus) across much of its range, as has competition with channel catfish (I. punctatus). Hybridization between headwater catfish and channel catfish also poses a threat to the headwater catfish. Our analyses of cytochrome-b sequences of headwater catfish from the Frio River, Devils River, and Independence Creek indicated that a population of headwater catfish occurs in the Frio River of the Nueces Drainage, where it was considered extirpated since 1967, and that hybridization is occurring in populations that inhabit the Frio River and Independence Creek. No obvious sign of hybridization was present in the population of headwater catfish in the Devils River.