Abstract: 36 hybrid combinations were obtained based on six tobacco cultivars Zhongyan90 (high resistance), Gexin3 (high resistance), L8 (middle resistance), Burley21 (middle resistance), KBM20 (high susceptibility) and Ky14 (high susceptibility) by the first method of C=P*P of Griffing diallel analysis. The combining ability of resistance to tobacco black shank were analyzed by DPS software. Results showed that there was great significant resistance difference among combinations. The resistance of different parents could be exhibited stably. General combining ability (GCA) and special combining ability (SCA) of resistance to black shank of six cultivars differed significantly. Zhongyan90 and KBM20 respectively showed the first negative and positive effect which indicated that Zhongyan90 could be better than KBM20 in resistance breeding. There was some difference of GCA among six parents. Zhongyan90 and Gexin3 used as high resistance parents could exhibit steady GCA when combined with Burley21. However, Ky14 and KBM20 had low GCAs and were not appropriate parents resistant to black shank. The combination of Burley21 and KBM20 with a high SCA should be further studied. Furthermore, dominant effect was the main part in the heritance of black shank resistance also with large additive effect. Meanwhile, environmental factors will affect resistance expression to some extent. The resistance of parents had one great influence on combinations though there was a stably heritance. As a result, it is not suitable to select objective lines in the early generation.