Fishes of the genus Prochilodus are ecologically and commercially important, ubiquitous constituents of large river biota in South America. Recent ecologic and demographic studies indicate that these fishes exist in large, stable populations with adult census numbers exceeding one million individuals. Abundance data present a stark contrast to very low levels of genetic diversity (Θ) and small effective population sizes (N_e) observed in a mitochondrial (mt) DNA dataset obtained for two species, Prochilodus mariae, and its putative sister taxon, Prochilodus rubrotaeniatus. Both species occupy major river drainages (Orinoco, Essequibo, and Negro) of northeastern South America. Disparity between expectations based on current abundance and life history information and observed genetic data in these lineages could result from historical demographic bottlenecks, or alternatively, natural selection (i.e., a mtDNA selective sweep). To ascertain underlying processes that affect mtDNA diversity in these species we compared Θ and N_e estimates obtained from two, unlinked nuclear loci (calmodulin intron-4 and elongation factor-1α intron-6) using an approach based on coalescent theory. Genetic diversity and N_e estimated from mtDNA and nuclear sequences were uniformly low in P. rubrotaeniatus from the Rio Negro, suggesting that this population has encountered a historical bottleneck. For all P. mariae populations, Θ and N_e based on nuclear sequences were comparable to expectations based on current adult census numbers and were significantly greater than mtDNA estimates, suggesting that a selective mtDNA sweep has occurred in this species. Comparative genetic analysis indicates that a suite of evolutionary processes involving historical demography and natural selection have influenced patterns of genetic variation and speciation in this important Neotropical fish group.