A list of mosquitoes from the Nakai Nam Theun National Protected Area along the Nam Theun, Nam Mon, Nam Noy, and Nam On rivers, Nakai District, Khammuane Province, Lao People's Democratic Republic (Lao PDR) is presented. Fifty-four mosquito taxa were identified, including 15 new records in the Lao PDR. A fragment of the mtDNA cytochrome c oxidase subunit I (COI) gene, barcode region, was generated for 34 specimens, and together with four specimens already published, it represented 23 species in eight genera. In addition, an updated checklist of 170 mosquito taxa from Lao PDR is provided based on field collections from Khammuane Province, the literature, and specimens deposited in the Smithsonian Institution, National Museum of Natural History (SI-NMNH), Washington, DC, U.S.A. This paper provides additional information about the biodiversity of mosquito fauna in Lao PDR.