A comparative linkage map for Culex tritaeniorhynchus was constructed based on restriction fragment length polymorphism markers using cDNAs from Aedes aegypti. Linear orders of marker loci in Cx. tritaeniorhynchus were identical to Culex pipiens wherein chromosomes 2 and 3 reflect whole-arm rearrangements compared to A. aegypti. However, the sex determination locus in Cx. tritaeniorhynchus maps to chromosome 3, in contrast to Cx. pipiens and Ae. aegypti where it is located on chromosome 1. Our results indicate that insensitive acetylcholinesterase (AChE)-mediated organophosphate resistance is controlled by a single major gene (AChE) on chromosome 2, while the AChE structural gene (Ace) is located on chromosome 1. No evidence for a second Ace gene was observed, even under very low stringency hybridization conditions.