The invention relates to molecular marker primers, and application thereof in early identification of seed sources of tobacco varieties. The molecular marker primers are trnH1/psbA1; trnH2/psbA2; trnH3/psbA3; and trnH4/psbA4. The molecular marker primers are applied to early identification of seed sources of tobacco varieties. The markers have the beneficial effects that any one pair of the four pairs of primers can be used for distinguishing and identifying conventional tobacco seeds and sterile line seeds in the prior art, that is, molecular marking of the conventional tobacco seeds and the sterile line seeds is completed, so that strict management and control of the tobacco seeds are realized. By using the method disclosed by the invention, distinguishing can be carried out by naked eyes, so that the sterile line tobacco species can be identified.