Abstract: It is efficient measure in controlling tobacco bacterial wilt to take full advantage of resistant germplasm and breeding resistant varieties. The objective of evaluating its combining ability is to improve breeding efficiency. Sixteen of tobacco germplasm, including different resistant to bacterial wilt and its F₁ cross by diallel mating design were identified in disease field of Anhui province in China in 2010 & 2011. The results showed that the general combining ability of tobacco resistant to bacterial wilt among varieties was significant difference, RG11, NC86, C17, Sheyuan 4 had the highest effect, and K346, Yanyan 97, K326, LMFC34 were the next. The specific combining ability was not significant difference. As a result, the screening of crosses among parents in resistance breeding program could be according to its highest resistance to tobacco bacterial wilt.