A entomopathogenic ascomycetous fungus, Cordyceps militaris is distributed world wide and cultivated as a medicinal mushroom having many valuable biological and pharmaceutical activities. In the breeding of new C. militaris mushroom, single ascospores were isolated and tested their characteristics such as mycelial growth, mycelial density, fruiting body formation ability, the production of perithecia. Also two opposite mating types were assayed using two sets of primers specific for C. militaris, which were amplified a 191-bp fragment for MAT1-1 and 233-bp fragment for MAT1-1. F1 hybrids resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the PCR assays. New hybrids were produced showing high quality fruiting bodies in artificial media. Mycelial growth of new strain 'Dowonhongcho 2’ was better on SDAY and at 15-25°C when it was compared with that of 'Yedang 3'. The stromata of new strain were club-shaped and bright orange-red color. Its length was 7.1 cm and the cordycepin content was 0.33% on average. In comparison with 'Yedang 3', the new strain had a yield that was 7% higher and it produced fruiting bodies which were firmer. Days of fruiting body were similar with 50 days from inoculation. This cultivar may serve as a valuable one for artificial cultivation and industrial-scale production of C. militaris.