The pink dolphin (Inia geoffrensis) is widely distributed along the Amazon and Orinoco basins, covering an area of approximately 7 million km2. Previous morphological and genetic studies have proposed the existence of at least two evolutionary significant units: one distributed across the Orinoco and Amazon basins and another confined to the Bolivian Amazon. The presence of barriers in the riverine environment has been suggested to play a significant role in shaping present-day patterns of ecological and genetic structure for this species. In the present study, we examined the phylogeographic structure, lineage divergence time and historical demography using mitochondrial (mt)DNA sequences in different pink dolphin populations distributed in large and small spatial scales, including two neighbouring Brazilian Amazon populations. mtDNA control region (CR) analysis revealed that the Brazilian haplotypes occupy an intermediate position compared to three previously studied geographic locations: the Colombian Amazon, the Colombian Orinoco, and the Bolivian Amazon. On a local scale, we have identified a pattern of maternal isolation between two neighbouring populations from Brazil. Six mtDNA CR haplotypes were identified in Brazil with no sharing between the two populations, as well as specific cytochrome b (cyt b) haplotypes identified in each locality. In addition, we analyzed autosomal microsatellites to investigate male-mediated gene flow and demographic changes within the study area in Brazil. Data analysis of 14 microsatellite loci failed to detect significant population subdivision, suggesting that male-mediated gene flow may maintain homogeneity between these two locations. Moreover, both mtDNA and microsatellite data indicate a major demographic collapse within Brazil in the late Pleistocene. Bayesian skyline plots (BSP) of mtDNA data revealed a stable population for Colombian and Brazilian Amazon lineages through time, whereas a population decline was demonstrated in the Colombian Orinoco lineage. Moreover, BSP and Tajima's D and Fu's Fs tests revealed a recent population expansion exclusively in the Bolivian sample. Finally, we estimated that the diversification of the Inia sp. lineage began in the Late Pliocene (approximately 3.1 Mya) and continued throughout the Pleistocene. Published 2011. This article is a US Government work and is in the public domain in the USA. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102, 812–827.