In Southeast Asia, despite the use of Japanese encephalitis vaccines and vaccination coverage, Japanese encephalitis (JE) transmission is still a major public health issue. The main vectors of this virus are mosquitoes from the genus Culex, which diversity and density are important in Southeast Asia. The main vector species of Japanese encephalitis virus (JEV) in Cambodia belong to the Vishnui subgroup. However, their morphological identification solely based on the adult stage remains challenging, making their segregation and detection difficult. In order to identify and describe the distribution of the three main JEV vector species in Cambodia, namely Culex vishnui , Cx. pseudovishnui and Cx. tritaeniorhynchus , mosquito samplings were carried out throughout the country in different environments. Phylogenetic analysis of the cytochrome c oxidase subunit I (coI) gene using maximum‐likelihood tree with ultrafast bootstrap and phylogeographic analysis were performed. The three main Culex species are phylogenetically separated, and represent two distinct clades, one with Cx. tritaeniorhynchus and the second with Cx. vishnui and Cx. pseudovishnui , the latter appearing as a subgroup of Cx. vishnui . The phylogeographic analysis shows a distribution of the Vishnui subgroup on the entire Cambodian territory with an overlapped distribution areas leading to a sympatric distribution of these species. The three JEV vector species are geographically well‐defined with a strong presence of Cx. pseudovishnui in the forest. Combined with the presence of Cx. tritaeniorhynchus and C x. vishnui in rural, peri‐urban, and urban areas, the presence of JEV‐competent vectors is widespread in Cambodia.