Abstract: To detect Phomopsis sp. in tobacco rapidly and accurately, PCR amplification, fragment recovery and DNA sequencing were carried out on the genomic DNA of Phomopsis sp. and 10 other tobacco pathogens by using fungal rDNA-ITS universal primers. Two pairs of specific detection primers were designed, and the detection sensitivity and specificity of the primers were evaluated. Furthermore, the PCR reaction system was optimized. The results showed that at the annealing temperature of 60 ℃, two pairs of specific primers P2F/P3R and P3F/P3R amplified specific bands of 420 bp and 381 bp from Phomopsis sp. However, no band was amplified in the other 10 tobacco pathogen DNAs and the negative controls. The detection sensitivity of both pairs of the specific primers reached 10 pg/µL. The established molecular detection method based on two pairs of specific primers P2F/P3R and P3F/P3R can rapidly and accurately detect Phomopsis sp. in tobacco.