Colletotrichum circinans, an anthracnose pathogen, causes serious damage to onions worldwide. In this study, specific molecular markers were developed to detect C. circinans 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 C. circinans, were constructed by analyzing tef-1α 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 C. circinans 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 C. circinans developed in this study may enhance the efficiency of fungal pathogen detection in imported vegetables and seeds.