High insecticide resistance resulting from insensitive acetylcholinesterase (AChE) has emerged in mosquitoes. A single mutation (G119S of the ace-1 gene) explains this high resistance in Culex pipiens and in Anopheles gambiae. In order to provide better documentation of the ace-1 gene and the effect of the G119S mutation, we present a three-dimension structure model of AChE, showing that this unique substitution is localized in the oxyanion hole, explaining the insecticide insensitivity and its interference with the enzyme catalytic functions. As the G119S creates a restriction site, a simple PCR test was devised to detect its presence in both A. gambiae and C. pipiens, two mosquito species belonging to different subfamilies (Culicinae and Anophelinae). It is possibile that this mutation also explains the high resistance found in other mosquitoes, and the present results indicate that the PCR test detects the G119S mutation in the malaria vector A. albimanus. The G119S has thus occurred independently at least four times in mosquitoes and this PCR test is probably of broad applicability within the Culicidae family.