The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. We report here the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 (5,879 bp), which share sequence identities of 62 and 95% at the 3'- and 5'-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were detected. The protein encoded by the 3'-proximal coding region of ORF I shows sequence identities of 19 to 23% with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae, Chrysoviridae, and Megabirnaviridae. However, the proteins encoded by the 5'-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of ~35 nm in diameter encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80, and 85 kDa, respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite dsRNA virus that possibly belongs to a new virus family.