Abstract: OBJECTIVE To investigate the difference in the content of active ingredients in toad venom of Bufo bufo gargarizans from different geographic locations, and analyze the regional characteristics of the Bufonis Venenum. METHODS The contents of 7 main active compounds were determined by HPLC, and the geographic location discipline of the samples were analyzed by hierarchical cluster analysis and radar chart. RESULTS The contents of bufogenin in different geographic locations was significant different. The dominant ingredients of the Bufonis Venenum from Eastern were the cinobufagin and resibufogenin, the total content ranged from 6.95% to 11.15%, while the contents in Central and Southwestern China ranged from 0.17% to 4.17%. The dominant ingredients of the Bufonis Venenum from Central and Southwestern China were the telocinobufagin and cinobufotalin, the total content ranged from 5.66% to 11.37%, while the contents in Eastern ranged from 1.22% to 2.26%. CONCLUSION The quality of Bufonis Venenum raw materials is mainly affected by the factors of geographic location. The results provide an important reference for improving the quality control of Bufonis Venenum.