Pleistocene environmental changes were important drivers of species- and population-level diversification in Anopheles mosquitoes. However, Anopheles species have different ecologies, so their response to Pleistocene cli- mate oscillations should have differed. We investigate whether genetic diversification in Anopheles punctimacula s.s. (a forest specialist and secondary vector of Plasmodium vivax) and Anopheles albimanus (a habitat colonist and primary vector of P. vivax and P. falciparum) is due to: (1) historical population processes, (2) contemporary population processes or (3) a combination of both. Differences in the degree of refugial isolation during the Last Glacial Maximum (LGM) and the degree to which isolated populations evolved habitat niche differences appear to explain differences in the phylogeographical patterns between A. punctimacula s.s. and A. albimanus in Lower Middle America (Mesoamerica). Refugial isolation during the LGM and subsequent niche diversification shaped the phylogeographical history of A. punctimacula s.s. During the LGM, the genetic pool of this species was fragmented into extremely narrow and scattered habitat refugia, resulting in two discrete mitochondrial lineages. Subsequently, these lineages appear to have further evolved distinct niche preferences and diversified due to different climatic conditions between populations, which may have contributed to the lack of introgression or range overlap among mitochondrial lineages. While A. albimanus also experienced range contraction, recovery was more rapid, and we find no evidence of niche evolution among lineages. This appears to explain the broad mitochondrial introgression in this species. Greater resilience to climatic instability by A. albimanus might contribute to its principal transmission role for human Plasmodium parasites across the Neotropics.