Abstract: In order to identify five tobacco root and stem disease pathogens, including Fusarium oxysporum, Phytophthora nicotianae, Rhizoctonia solani, Thieleaviopsis basicola, and Aphromyces iridis rapidly and accurately, specific amplification primers were screened and designed. The primer concentration, annealing temperature and number of cycles of the reaction system were optimized to establish a multiple PCR detection system, and its practicability in detecting pathogens was verified. The specific primer pairs of LD141 F/R, YM1002 F/R, LK111 F/R, GHFT F/R, AiT7 F/R for F. oxysporum, P. nicotianae, R. solani, T. basicola and A. iridis were screened by RAPD molecular markers and designed. The optimal primer concentration in the PCR reaction system was 1.0, 1.0, 0.2, 0.5, 0.5 μmol/L, respectively, and the optimal annealing temperature was 54 ℃, the number of optimal cycles was 28. The specific fragments with sizes of 370, 240, 536, 783, 138 bp could be amplified simultaneously and the detection sensitivity of five pathogens DNA reached 0.5 ng/μL. The detection system could quickly detect F. oxysporum, P. nicotianae, R. solani, T. basicola and A. iridis in seedling substrate samples and infected tobacco plants, which might be useful for early prevention of tobacco root and stem diseases.