, an anthracnose pathogen, causes serious damage to onions worldwide. In this study, specific molecular markers were developed to detect accurately and quickly with both conventional and real-time PCR methods. The cirTef-F/cirTef-R and cirTu-F/cirTu-R primer sets, which are specific for , were constructed by analyzing and β-tubulin genes in the fungus. Using the conventional PCR method, 100 pg and 1 ng of fungal DNA could be detected using the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Using the real-time PCR method, 10 pg and 100 pg of fungal DNA could be detected more sensitively with the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Detection of from the artificially infected onion seeds was possible by using both conventional and real-time PCR methods and the developed cirTef-F/cirTef-R primer set. The PCR markers specific for developed in this study may enhance the efficiency of fungal pathogen detection in imported vegetables and seeds.