In specific altitude ranges, biotic and abiotic factors can impact vector mosquitoes’ adaptation capacity, affecting their population differentiation. This study analyses if there exist morphological and genetic differences in four Anopheles (Kerteszia) populations in specific altitude ranges from the Colombian pacific coast to the premontane humid forests in Valle del Cauca, Colombia. Likewise, it is compared if the vector mosquito groups analyzed were genetically similar to the ones available in the region. Traditional and geometric morphometric analysis and the molecular marker CO-I were used. The research found that vector mosquitoes’ littoral populations differentiated morphologically according to their cross veins wing shapes compared to the other three groups in higher altitudes. Their genetic distances fluctuate between 4.95% and 6.84%, indicating that vector mosquitoes’ littoral populations belong to Anopheles neivai s.s. while the ones of higher altitudes are related to An. neivai 8—a lineage previously proposed based solely on molecular data. The study concludes that vector mosquitoes at the pacific Colombian coast from the littoral area in lower altitudes maintain a vast genetic variability with uniform populations; however, in higher altitudes, vector mosquitoes acquire molecular and morphological differences that may include the settlement of other lineages.