Phylogeography and intraspecific genetic variation were studied in prochilodontids endemic to the Orinoco, Essequibo and Amazon River basins of northern South America. Portions of two protein-encoding mitochondrial (mt) DNA genes, ND4 and COI, were examined using single-strand conformational polymorphisms (SSCPs) and nucleotide sequencing. Phylogeographic analysis indicated that the geographically widespread Prochilodus rubrotaeniatus is paraphyletic, with individuals from the Orinoco sharing most recent common ancestry with co-occurring Prochilodus mariae. A second Prochilodus rubrotaeniatus clade was composed of haplotypes found in the Rio Cuyuní(Essequibo Basin) and tributaries of the Rio Negro (Amazon Basin). Intraspecific genetic analysis suggested that a complex set of processes have influenced patterns of genetic variation in prochilodontid lineages. Prochilodus rubrotaeniatus is monomorphic at both loci in the Rio Negro and probably recently colonized this basin from the Rio Essequibo. Only two of 55 P. mariae exhibited variant haplotypes, and both had resulted from non-synonymous changes in the ND4 region. These observations were counter to neutral expectation and consistent with the action of natural selection on the mitochondrion. Overall, these analyses implicate vicariance, demography and selection for driving diversification of prochilodontids in northern South America.