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<p>Abstract</p> <p>Background</p> <p>Carboxylesterase overproduction is a frequently observed resistance mechanism of insects to organophosphate insecticides. As a major transmitter of human diseases, mosquitoes in the <it>Culex pipiens</it> complex have evolved 13 carboxylesterase alleles (<it>Ester</it>) that confer organophosphate resistance. Six alleles, <it>Ester</it>^<it>B1</it>, <it>Ester</it>^<it>2</it>, <it>Ester</it>^<it>8</it>, <it>Ester</it>^<it>9</it>, <it>Ester</it>^<it>B10</it>, and <it>Ester</it>^<it>11</it>, 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.</p> <p>Results</p> <p>Based on the DNA sequences of resistant and nonresistant carboxylesterase alleles, <it>Ester B</it> alleles were first amplified with PCR-specific primers and then digested with the restriction enzyme <it>Dra</it>I. In this step, <it>Ester</it>^<it>2</it> and <it>Ester</it>^<it>11</it> were differentiated from the other <it>Ester</it> alleles. When the other <it>Ester</it> B alleles were digested with the restriction enzyme <it>Xba</it>I, <it>Ester</it>^<it>B1</it> and the susceptible <it>C. p. pallens Ester</it> were screened out. <it>Ester</it>^<it>8</it> and <it>Ester</it>^<it>9</it> were differentiated from <it>Ester</it>^<it>B10</it> and the susceptible <it>C. p. quinquefasciatus</it> esterase allele, respectively, by amplifying and digesting the <it>Ester A</it> alleles with the restriction enzyme <it>Apa</it>LI. The effectiveness of the custom-designed PCR-RFLP was verified in two field mosquito populations.</p> <p>Conclusions</p> <p>A PCR-RFLP based approach was developed to differentiate carboxylesterase alleles in <it>Culex pipiens</it> 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.</p>