evidence: Given the existing drastic variations of malaria conditions in Cambodia, this work aimed to study the mosquito diversity and molecular phylogeny of secondary vectors i.e., An. nivipes in the Cambodia-Laos border. Siem Pang County (Stung treng Province) was taken as the investigation site and mosquito surveillance was firstly performed. The cytochrome c oxidase subunit 2 gene (COII) of mtDNA from An. nivipes samples were subsequently sequenced. Furthermore, the gene variability and phylogenetic relationship of An. nivipes along the Cambodia-Laos border were investigated and compared with other samples from India, Thailand, and Myanmar for examining their gene relations. This work largely aimed at gaining more insights into the continuous surveillance of mosquito vectors, as well as molecular phylogeny and evolution of An. nivipes in the Cambodia-Laos border by using an effective genetic marker, mtDNA-COII. Stung treng borders Laos in the north and is located on the east bank of the Mekong River. This endemic region takes up nearly 11,000 square kilometres and has about 42,000 residents. The average population density is 3.82 people per square kilometre. It has dense forests, diversified landforms, the temperature changes greatly and the annual rainfall is 1800 mm.