Abstract: To investigate the physiological race classification and pathogenicity regarding main cultivars of Phytophthora parasitica var. nicotianae in Yuxi tobacco-growing areas of Yunnan, 172 strains of P. parasitica var. nicotianae were isolated from Xinping County and Chengjiang City in Yuxi. Their physiological races and group distribution were identified with TTC method and in vivo plant inoculation method. Using P. parasitica var. nicotianae strains of different physiological races as inoculums, the inoculation technology for physiological race identification was studied, and its reliability was verified. The results showed that the tested strains were magenta on TTC plate and belonged to physiological race 0 or 1. In vivo plant inoculation results indicated that there were 149 strains of race 0, 12 strains of race 1, 2 non-pathogenic strains, and 9 unidentified strains, accounting for 86.63%, 6.98%, 1.16%, and 5.23%, respectively, which suggested that race 0 was the main physiological race of P. parasitica var. nicotianae in Xinping County and Chengjiang City. The pathogenicity test results showed that 170 strains had strong pathogenicity to the main cultivars KRK26, Yunyan 87 and K326. In addition, the resistant or susceptible reaction of slightly injured in vitro leaves and stalks of the host to the physiological race of P. parasitica var. nicotianae was consistent with that of in vivo plants after inoculation injection, and they had equivalent discriminability. In conclusion, the wound inoculation method of in vitro materials can be used to distinguish the resistance of different tobacco varieties to black shank disease.