【Abstract】 Objective To explore the value of isozyme zymograms in the classification of blackflies and accumulate basic biochemical data for the study of phylogenesis. Methods EST isozymes of three blackfly species were studied by discontinuous PAGE techniques. Results Ten isozyme bands could be identified according to the electrophoresis zymograms. The differences of zymograms were significant between three blackfly species and each species had its own special zymogram. The main band among intraspecific individuals or between the larvae and pupae was alike， however， other enzyme bands were different. Conclusion EST isozyme zymograms could be used as one of the classification characteristics of blackflies.