Abstract: In order to investigate population structure and metalaxyl resistance level of Phytophthora nicotianae on tobacco black shank disease, the methods of confrontation culture and fungicide-amended plate were used to determine mating type and sensitivity to metalaxyl of P. nicotianae isolates collected from 2013 to 2014 in Dali, Yunnan Province. The results showed that all 150 isolates of P. nicotianae from 12 counties were A2 mating type and A1 or A0 mating type was not identified. The EC₅₀ values of a total of 263 isolates to metalaxyl distributed between 1.0829 and 33.6227 mg/L, with an average EC₅₀ value of 8.7599±0.0358 mg/L. The ratio of sensitive, moderate resistant and resistant population was 14.83%, 52.85% and 32.32%, respectively, which means that sensitivity to metalaxyl of P. nicotianae isolates collected from Dali had significant differences. Our results suggest that the population structure of P. nicotianae was mainly maintained through asexual propagation in its life cycle in Dali. The pathogen had a high resistance level to metalaxyl and both moderate and resistant isolates were widely distributed. It is urgent to screen and make use of new high-efficiency and safe alternatives for black shank disease management.