Abstract: ObjectiveThe present study was conducted to select the most suitable culture conditions and optimal cul-ture media for culturing primary cells of eggs(embryos) in Simulium(Wilhelmia) xingyiense, and identify the primary cells in order to lay a foundation for establishment of blackfly cell lines. MethodPrimary cells of eggs(embryos) in S.(W.) xingyiense were cultured using tissue block method and single cell method. The influence factors for cell culture were ana-lyzed, including medium, temperature, pH value, CO2 and culture vessels. At the same time, the sequences of rDNA-ITS region of eggs(embryos) and primary cells were determined, and blackfly phylogenetic tree was established based on their ITS sequences to identify their species origin . ResultThe tissue block method was the optimal culture method for culturing the primary cells of eggs(embryos) in S.(W.) xingyiense. The appropriate culture medium was modified Shields and Sang M3 with pH 6.0-6.5 and 28℃culture temperature. Constant temperature biochemical incubator and 5%CO2 in-cubator made no significant differences on culturing primary cells. Poly-L-lysine(PLL) treated plastic petri dishes/plates could be used as culture vessels. The result of blackfly phylogenetic tree based on ITS sequences showed that blackfly eggs (embryos) and their primary cells were derived from S.(W.) xingyiense. ConclusionThe culture method for primary cells of eggs(embryos) in S.(W.) xingyiense successfully established can be further applied in scientific research and production.