Recently, the introduction and afforestation of the poplar species were increased and the risks of pests and diseases carried by poplar were highly increased. Among the diseases, poplar rust caused by Melampsora larici-populina, is the main threaten to poplar sapling and seedling. This pathogen can damage leaves, petioles, young shoots, and winter buds. The disease usually happens in seedlings, which causes risk on the introduction of exotic seedlings. However, the fungal pathogen is not yet cataloged in the BAPHIQ quarantine items. In this study, we extracted the nucleic acid of infected poplar samples from Tainan and accomplished the ITS sequence analysis. The ITS sequence showed that sequence homology was similar with the rust pathogen species of M. larici-populina published in China and Sweden regions. The pathogen is difficult to identify due to obligation parasite. Moreover, the external symptoms of the rust spores in host leaves are difficult to observe and identify in the early infection stage. In this study, the ITS region was amplified and sequenced, then three primer sets were designed to examine the specificity to M. larici-populina. The results of specificity test of three primer sets showed that the F1/R1 primer pairs posses the best specificity and high sensitivity, the pathogen nucleic acid could still be detected under 1.5 pg.