The Neotropical malaria vector Anopheles aquasalis Curry is distributed predominantly along the Atlantic and Pacific Coasts because of its tolerance for breeding in salt water. We tested the hypothesis that the freshwater Amazon River acts as a barrier to gene flow in northeastern Brazil, by examining variation at a 588-nucleotide fragment of the mitochondrial cytochrome oxidase I gene from five populations. We identified 15 haplotypes of which 5 were shared both (1) between sample localities and (2) across the Amazon River Delta. Sequence divergence ranged from 0.0017–0.0272 (average = 0.0137). Estimates of genetic subdivision based on the presence of the Amazon River were greatest within localities (Φ = 0.029) and among regions (Φ = 0.018), followed by among localities (Φ = 0.011), but none were significant. Parsimony, neighbor-joining, and Nested Clade Analyses were used to estimate relationships among populations and infer evolutionary processes. Two phylogenetically distinct clusters of populations were moderately supported by parsimony. Neighbor-joining trees were poorly resolved, thus providing no geographical resolution and no support for the Amazon River as a barrier to migration. Phylogeographic structure as detected by the Nested Clade Analysis was consistent with restricted gene flow coupled with isolation by distance. Taken together, these analyses suggest that the localities within this region of northeastern Brazil constitute a single large population of An. aquasalis that spans the Amazon Delta.