Abstract: To clarity the pathogens of tobacco Fusarium root rot and their pathogenicity, samples showing root rot symptoms from Zhuji, Shixing, Nanxiong and Wujing in Guangdong were collected. Strains were isolated by tissue separation and their pathogenicity to tobacco seedlings was determined by inoculation of wheat grains with fungi in potted tobacco plants. The strains were identified based on morphological characteristics, combined with the phylogenetic analysis method using partial sequences of EF-1α and β-Tub. 45 Fusarium strains were isolated. The results of morphological and molecular biological identification showed that 45 strains belonged to different species, i.e., Fusarium solani, F. commune, F. fujikuroi and F. oxysporum, and they accounted for 42.22%, 26.67%, 20.00% and 11.11%, respectively. They all had pathogenicity to tobacco, and the order of pathogenicity was F. commune≈F. fujikuroi>F. oxysporum>F. Solani. Identification of pathogens of tobacco Fusarium root rot may help in establishing the appropriate measures to control this disease.