Pathogenic and genotypic variability among seven populations of Phytophthora parasitica var. nicotianae from individual tobacco fields in Qujing, Kunming, Honghe, Chuxiong, Yuxi, Dali of Yunnan province and Shandong were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses. About 6 strains were isolated from different fields in Honghe, 11 and 12 strains from different fields in Kunming and Yuxi , 9 strains from Qujing, and 9 and 8 stains from Chuxiong and Dali, another 5 strains from Shandong province, respectively, using tobacco cultivar (Hongda) as a baiting host. All 60 strains belonged to race 1 in this study. Pathogenic variability was evaluated in greenhouse studies with five tobacco cultivars which have different levels of resistance to black shank of tobacco. All strains were pathogenic on one or more of five tobacco cultivars. Disease severity incited by different strains varied significantly on individual tobacco cultivar. The percentage of strains pathogenic on different cultivars varied among isolations. Genotypic variation among 60 strains was evaluated with RAPD analysis. The selected primers detected 70 polymorphic bands. Cluster and principal coordinates analysis revealed one main cluster and one minor cluster, the main cluster group containing 48 of the 60 strains from all locations, the minor cluster group containing 12 of the 60 strains from Yunnan, and the strains with about 89% similarity to the main groups of 48 strains. The strains in the minor group were all derived from different regions of Yunnan province and were pathogenic on three tobacco cultivars (NC89, NC82 and Hongda), the main strains were pathogenic (DS = 2.0) on three cultivars (NC89, NC82 and Hongda), too. The result showed that P. parasitica var. nicotianae populations were genotypically and phenotypically variable, and the polymorphic markers were not associated with the pathogenic variation.