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Oyster mushroom spherical virus (OMSV) is a mycovirus with a positive-sense single-stranded RNA genome that infects the edible mushroom <i>Pleurotus ostreatus</i>. 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 from <i>P. ostreatus</i> to <i>Pleurotus pulmonarius</i>. However, transmission was not successful in other <i>Pleurotus</i> species including <i>P. citrinopileatus</i>, <i>P. eryngii</i>, <i>P. nebrodensis</i>, and <i>P. salmoneostramineus</i>. The successful OMSV infection in <i>P. pulmonarius</i> was 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 of <i>P. pulmonarius</i>. The OMSV-infected strain demonstrated abnormal performance including twisted mushrooms or irregular edge of the cap as well as reduced yield of fruiting bodies in <i>P. pulmonarius</i>, compared to the OMSV-free strain. This study is the first report on the infection and pathogenicity of OMSV to the new host <i>P. pulmonarius</i>. The data from this study therefore suggest that OMSV is a potential threat to <i>P. pulmonarius</i>.