Abstract:The technique of molecular detection of Callosobruchus analis (Fabricius) and Callosobruchus maculatus (Fabricius) was studied using the methods of polymerase chain reaction and enzyme digestion. According to the alignments of mtDNA COⅠ gene sequences of bean weevils, two specific primers were designed that the optimal annealing temperature for PCR was 55℃ and two restriction enzymes were screened out for molecular detection. The results showed that different populations, individuals and consistence of C.analis and C.maculatus could obtain corresponding amplificatory bands by the specific primers and the results were testified by the enzyme digestion experiment. The PCR detection and enzyme digestion with high sensitivity and accuracy could make up for a lack of morphology.