Mosquitoes are the main arthropod vectors of infectious diseases in humans. The current methods for mosquito identification include morphological and molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), now routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of this study was to use MALDI-TOF MS to identify mosquito colonies from French Polynesia. Five hundred specimens from French Polynesia belonging to three species, Aedes aegypti, Aedes polynesiensis, and Culex quinquefasciatus, were included in the study. Testing the legs of these mosquitoes by MALDI-TOF MS revealed a 100% correct identification of all specimens at the species level. The MALDI-TOF MS profiles obtained allowed differentiation of male from female mosquitoes and the specific identification of female mosquito colonies of the same species but different geographic origin.