A novel positive single-stranded RNA virus, Pleurotus ostreatus deltaflexivirus 1 (PoDFV1), was isolated from the edible fungus Pleurotus ostreatus strain ZP6. The complete genome of PoDFV1 is 7706 nucleotides (nt) long and contains a short poly(A) tail. PoDFV1 was predicted to contain one large open reading frame (ORF1) and three small downstream ORFs (ORFs 2-4). ORF1 encodes a putative replication-associated polyprotein of 1979 amino acids (aa) containing three conserved domains – viral RNA methyltransferase (Mtr), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) – which are common to all deltaflexiviruses. ORFs 2-4 encode three small hypothetical proteins (15-20 kDa) without conserved domains or known biological functions. Sequence alignments and phylogenetic analysis suggested that PoDFV1 is a member of a new species in the genus Deltaflexivirus (family Deltaflexiviridae , order Tymovirales ). To our knowledge, this is the first report of a deltaflexivirus infecting P. ostreatus .