Raffaelea quercus‐mongolicae is a mycangial symbiont of a wood‐boring ambrosia beetle, Platypus koryoensis (Coleoptera: Curculionidae: Platypodidae), which is believed to be associated with oak mortality in South Korea. Since the first discovery of the disease in 2004, oak mortality has continued to spread across the country, and the pathogen has had a significant impact on forest ecosystems in the country; however, little is known regarding the biology of the fungus. The aim of this study was to develop markers to determine the mating types of individual isolates of R. quercus‐mongolicae collected across the country and then determine the mating‐type ratio in natural populations. To achieve this, partial sequences of MAT1‐1–3 and MAT1‐2–1 genes were amplified using degenerate primers and cloned. The new primer sets, which were specific to each mating‐type idiomorph, identified isolates as MAT1‐1 and MAT1‐2 mating types. The roughly equal frequencies of the two mating types suggest that sexual reproduction might have occurred in natural populations of R. quercus‐mongolicae in South Korea.