Abstract: As the mutant pool of T-DNA insertion has been constructed, a quantity of T-DNA flanking sequences are in great need to be cloned. TAIL-PCR, PCR-walking and FPNI-PCR have been used to clone the sequences flanked by known sequences. For this reason, it will be great help to choose an efficient method applied to the analysis of the tobacco T-DNA insertion flanking sequences. This study focus on the comparison among the efficiency, T-DNA insertion localization and the researched homologous protein of the three methods, also we optimized the PCR procedures and primers to select an effective method which is suitable for cloning of tobacco T-DNA flanking sequences. By comparing the efficiency, T-DNA insertion localization and the researched homologous protein, the result of TAIL-PCR was the best. This result lays a foundation for the study on tobacco gene function by using mutant.