The evolutionary history of Amazonian organisms is generally poorly understood. This is particularly true for small floodplain fish species that show reduced dispersal capabilities. The one-lined pencilfish Nannostomus unifasciatus (family Lebiasinidae) is a small fish found in flooded forests of the Rio Negro Floodplain (RNF) in central Amazonia, Brazil. We used a large number of samples collected throughout the species distribution in the RNF and in its headwaters and DNA data from the second intron of the S7 ribosomal protein to reconstruct the phylogeography of N. unifasciatus. Two markedly distinct phylogroups of N. unifasciatus were detected in the RNF. Although these lineages are largely allopatric, they remain reproductively isolated in regions where they overlap, suggesting cryptic speciation in this group in the Rio Negro basin. Coalescent-based statistical methods suggest that the history of these populations was dominated by a Miocene fragmentation of the species in the headwaters of the basin that originated the two phylogroups, followed by recent events of demographic and range expansions in the floodplain. This pattern is discussed within the context of the geomorphologic history of the region, especially geotectonics, and of marine incursions. Our results match the predictions of the palaeogeography hypothesis of speciation and outline the usefulness of an intron DNA marker to reconstruct population history of a central Amazonian fish. Because N. unifasciatus is harvested commercially in the ornamental fishery of the RNF, the two species and limited dispersal capacity between nearby populations identified here are also important to develop sound management decisions in the region.