Between 2016 and 2020, the Medical and Veterinary Entomology unit of the Institut Pasteur du Cambodge collected over 230,000 mosquitoes. Based on this sampling effort, a checklist of 290 mosquito species in Cambodia is presented. This is the first attempt to list the Culicidae fauna of the country. We report 49 species for the first time in Cambodia. The 290 species belong to 20 genera: Aedeomyia (1 sp.), Aedes (55 spp.), Anopheles (53 spp.), Armigeres (26 spp.), Coquillettidia (3 spp.), Culex (57 spp.), Culiseta (1 sp.), Ficalbia (1 sp.), Heizmannia (10 spp.), Hodgesia (3 spp.), Lutzia (3 spp.), Malaya (2 spp.), Mansonia (5 spp.), Mimomyia (7 spp.), Orthopodomyia (3 spp.), Topomyia (4 spp.), Toxorhynchites (4 spp.), Tripteroides (6 spp.), Uranotaenia (27 spp.), and Verrallina (19 spp.). The Cambodian Culicidae fauna is discussed in its Southeast Asian context. Forty-three species are reported to be of medical importance, and are involved in the transmission of pathogens.