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Background Carboxylesterase overproduction is a frequently observed resistance mechanism of insects to organophosphate insecticides. As a major transmitter of human diseases, mosquitoes in the Culex pipiens complex have evolved 13 carboxylesterase alleles (Ester) that confer organophosphate resistance. Six alleles, Ester B1 , Ester 2 , Ester 8 , Ester 9 , Ester B10 , and Ester 11 , have been observed in field populations in China, sometimes co-existing in one population. To differentiate the carboxylesterase alleles found in these field populations, PCR-RFLP was designed for use in resistance monitoring. Results Based on the DNA sequences of resistant and nonresistant carboxylesterase alleles, Ester B alleles were first amplified with PCR-specific primers and then digested with the restriction enzyme Dra I. In this step, Ester 2 and Ester 11 were differentiated from the other Ester alleles. When the other Ester B alleles were digested with the restriction enzyme Xba I, Ester B1 and the susceptible C. p. pallens Ester were screened out. Ester 8 and Ester 9 were differentiated from Ester B10 and the susceptible C. p. quinquefasciatus esterase allele, respectively, by amplifying and digesting the Ester A alleles with the restriction enzyme Apa LI. The effectiveness of the custom-designed PCR-RFLP was verified in two field mosquito populations. Conclusions A PCR-RFLP based approach was developed to differentiate carboxylesterase alleles in Culex pipiens complex mosquitoes. These processes may be useful in monitoring the evolutionary dynamics of known carboxylesterase alleles as well as in the identification of new alleles in field populations.