Abstract: 【Objective】To clarify species and biological characteristics of frog eye disease pathogen from flue-cured tobacco，so as to provide scientific basis for controlling flue-cured tobacco frog eye disease.【Method】 Flue-cured tobacco leaves with frog eye disease in Guizhou tobacco planting areas were taken as material.Pathogens were isolated and their pathogenicity was determined by conventional tissue isolation method and in vitro leaf inoculation method.Pathogens were classified and identified according to morphological characteristics and molecular biology methods.And the biological characteristics of typical strains were determined.【Result】Eleven strains of bacteria were isolated from collected diseased tobacco leaves，and 3 typical strains of YC1110，YC1111 and YC1112 were inoculated into healthy flue-cured tobacco.After six d of inoculation，tobacco leaves showed typical symptoms as the symptoms in field，proving the 4 strains were frog eye disease pathogen.ITS sequence and phylogenetic analysis showed that the 3 tested strains had close genetic relationship with Cercospora nicotianae，and the morphological characteristics of colonies and spores were also consistent with those of C.nicotianae.Biological characteristic determination showed that the highest spore yield required a temperature with was 25℃，pH of 7，24 h darkness of light condition，carbon and nitrogen sources of fructose and beef extract powder，the medium of tomato agar medium（TA2）.The optimum condition for the fastest growth were temperature of 25℃，pH of 7，carbon and nitrogen sources of mannitol and peptone，medium of carrot agar（CA）and tobacco leaf juice agar medium（TA1）.The lethal temperature of pathogen mycelia was 54℃ water bath for 10 min.【Conclusion】 The pathogen causing frog eye disease of flue-cured tobacco in Guizhou is identified as C.nicotiana.