Abstract: Tobacco is an important agricultural model plant that plays a significant role in the economies of many countries. Furthermore, tobacco is considered to be one of important model systems in plant biotechnology. However, the genetic studies, such as molecular marker development, linkage map construction and gene cloning of tobacco, are relatively backward. The reasons are most likely that tobacco is a tetraploid species with the biggest genome size in Solanaceae and complex genome structure. Therefore, it is important to develop excellent molecular markers for tobacco genetic study. Then, a new strategy for tobacco-specific TRAP molecular marker development was adopted in this study, in which the primers of tobacco EST-SSR was shared directly for the fixation primers of TRAP markers, and the first set of tobacco TRAP markers were developed. Compared with previous studies, the new strategy has several advantages, such as shorter process, lower cost and more polymorphism than EST-SSR. Moreover, the further studies confirm that the new strategy is useful for tobacco molecular genetic studies because of the good PCR amplification and a high level genetic diversity. In addition, a co-dominant heredity TRAP characteristic band of special aroma of oriental tobacco-like genotype was found, which can be transformed to SCAR marker or used in marker assisted selection (MAS) directly.