Sequences of the mitochondrial (mt) NADH dehydrogenase subunit 5 gene (ND5) were acquired to assess genetic diversity and female effective population size (Nef) of two forms of Cyprinella (C. lepida and C. sp. cf lepida) and two species of Dionda (D. serena and D. texensis) in headwaters of three rivers in the upper Nueces River basin in central Texas. As documented in prior studies, two divergent clades of haplotypes of mtDNA were found in both genera: one in the Frio and Sabinal rivers, representing C. lepida and D. serena; one in the Nueces River, representing C. sp. cf lepida and D. texensis. Levels of variation in mtDNA from C. lepida in the Sabinal River and D. serena in the Frio and Sabinal rivers were comparable to or considerably lower than values documented for populations of several threatened or endangered cyprinids. Estimates of Nef for C. lepida in the Frio River and C. sp. cf lepida in the Nueces River were low, suggesting that adaptive genetic variation through time may be compromised. Of all populations sampled, only D. texensis in the Nueces River appears at present to be genetically stable demographically. An unexpected finding was two individuals resembling C. lepida in the Frio River with a haplotype referable to C. sp. cf lepida; the origin of these individuals is unknown. Two other individuals resembling C. lepida but with haplotypes of mtDNA referable to C. venusta were found in the Frio River and presumably represent relatively recent hybrids. Results of our study indicate that C. lepida, C. sp. cf lepida, and D. serena in the upper Nueces River basin, especially in the Sabinal River drainage, are at appreciable genetic risk.