Abstract: ObjectiveThe present study investigated species and distribution of root-knot nematodes in tobacco grow-ing regions of Yunnan, in order to provide reference for comprehensive prevention of root-knot nematodes disease and to-bacco variety layout. MethodDuring August to October, 2015, 54 samples of root-knot nematode host on tobacco crops were collected from Kunming city, Qujing city and Yuxi city in Yunnan. The species of root-knot nematodes were identified by observing perineal patterns and molecular biological testing (PCR of ITS region of ribosomal DNA and sequence charac-terized amplified region , SCAR ) . ResultThere were 24 samples belonging to Meloidogyne arenaria, accounting for 44.44%;seven M. incognita samples, accounting for 12.96%;one M. javanica sample, accounting for 1.85%. Eleven sam-ples were compound population of M. javanica and M. arenaria, accounting for 20.37%. Five were compound population of M. incognita and M. arenaria, accounting for 9.26%. Three were compound population of M. incognita and M. javanica, ac-counting for 5.56%. Three belonged to compound population of M. incognita, M. javanica and M. arenaria, accounting for 5.56%. ConclusionThe species of root-knot nematodes in tobacco growing areas of Yunnan mainly include M. incognita, M. javanica and M. arenaria. M. arenaria is the dominant population.