Anopheles darlingi is the most important malaria vector in the Amazon Basin, and its recent resurgence in places such as Iquitos, Peru and Manaus, Brazil have been linked to increased malaria cases. Accurate identification of mosquito species and populations in susceptible geographical regions is critical for implementing effective vector control techniques. Molecular genetics and phylogeographic analysis can provide insight into the processes influencing the geographic dispersal of genetic lineages. To analyze the genetic relatedness and phylogeographic structure of A. darlingi from 19 localities throughout Central and South America, including Brazil, Peru, Belize, Colombia, French Guiana, and Guatemala, we used the nested clade analysis (NCA), and haplotype and nucleotide diversity with mitochondrial cytochrome oxidase subunit I (COI) sequences. The NCA results proposed that the current distribution of A. darlingi among the localities was attributable to the historical evolutionary process of contiguous range expansion, and the current process of restricted gene flow with isolation by distance. All of the A. darlingi populations had high haplotype diversity relative to nucleotide diversity, which suggests a population bottleneck followed by a rapid population expansion and buildup of mutations. Phylogeographic analyses can correlate the evolutionary history to the geographic distribution of a population. The ultimate goal is effective vector control techniques and possibly forecasting changes in malaria transmission patterns.