This study analyzed genetic differentiation of 20 Aedes aegypti populations collected along a street in Phnom Penh Municipality of Cambodia. Using allozyme and microsatellite variations, we demonstrated that populations were differentiated and the pattern of differentiation was dependent on the type of breeding sites. Moreover, insecticide treatments with temephos mostly affect the population functioning of discarded containers. Low gene flow detected could limit the natural diffusion of resistant populations that might instead take advantage of human displacements to spread.