By introducing purslane DNA into tobacco via pollen-tube pathway, the 4th generation self-bred progeny (D4) was obtained, and the purity of 32 selected D4 were analyzed. The results showed that the optimum SRAP-PCR reaction system includes a solution of 10 μL, which contains 0.2 mmol/L dNTP, 20 ng template DNA, 30 nmol/L primer, 0.5 U Taq polymerase and 2 mmol/L MgCl₂, annealing temperature 53 °C and cycling 35 times. The fingerprint of D4 was established by combining 118 selective primers, the DNA segment number ranged from 10 to 30 with a size of 100-700 bp in different primer combinations, a total of 1052 bands were amplified and 10 of the bands expressed polymorphism, which accounted for only 0.95%. It indicated that testing the purity of tobacco’s progeny with SRAP mark was feasible.