Oyster mushroom spherical virus (OMSV) is a mycovirus with a positive-sense single-stranded RNA genome that infects the edible mushroomPleurotus ostreatus. OMSV is horizontally transferred from an infected strain to a cured strain via mycelia. The infection results in significant inhibition of mycelial growth, malformation of fruiting bodies, and yield loss in oyster mushrooms. This study successfully transferred OMSV fromP. ostreatustoPleurotus pulmonarius. However, transmission was not successful in otherPleurotusspecies includingP. citrinopileatus,P. eryngii,P. nebrodensis, andP. salmoneostramineus. The successful OMSV infection inP. pulmonariuswas further verified with Western blot analysis using a newly prepared polyclonal antiserum against the OMSV coat protein. Furthermore, OMSV infection reduced the mycelial growth rate ofP. pulmonarius. The OMSV-infected strain demonstrated abnormal performance including twisted mushrooms or irregular edge of the cap as well as reduced yield of fruiting bodies inP. pulmonarius, compared to the OMSV-free strain. This study is the first report on the infection and pathogenicity of OMSV to the new hostP. pulmonarius. The data from this study therefore suggest that OMSV is a potential threat toP. pulmonarius.