A rapid, simple and accurate real-time PASA (PCR amplification of specific allele) (rtPASA) protocol was developed and optimized for the frequency estimation of the Glyl19Ser mutation in the type-l acetylcholinesterase locus, putatively associated with organophosphate resistance, in pooled DNA samples of Anopheles sinensis, a major vector mosquito of malaria in Korea. Performance of the rtPASA protocol was evaluated by comparing with the data generated from individual genotypings of a field population. The resistance allele frequency of the population (74.4%) predicted from the linear regression line of the rtPASA agreed well with that estimated from the individual genotyping (74.1%), demonstrating its reliability and accuracy. Using this rtPASA protocol, the resistance allele frequency in 10 local populations of An. sinensis was determined to range from 74.4% to 97.2%, suggestive of the widespread organophosphate resistance in An. sinensis in Korea.