Arbuscular mycorrhizal (AM) fungi that belong to the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. One of the four viral segments found by deep-sequencing of dsRNA in the AM fungus Rhizophagus clarus strain RF1 showed similarity to mitoviruses and is characterized in this report. The genome segment is 2,895 nucleotides in length, and the largest ORF was predicted by applying either the mold mitochondrial or the universal genetic code. The ORF encodes a polypeptide of 820 amino acids with a molecular mass of 91.2 kDa and conserves the domain of the mitovirus RdRp superfamily. Accordingly, the dsRNA was designated as R. clarus mitovirus 1 strain RF1 (RcMV1-RF1). Mitoviruses are localized exclusively in mitochondria and thus generally employ the mold mitochondrial genetic code. The distinct codon usage of RcMV1-RF1, however, suggests that the virus is potentially able to replicate not only in mitochondria but also in the cytoplasm. RcMV1-RF1 RdRp showed the highest similarity to the putative RdRp of a mitovirus-like ssRNA found in another AM fungus, followed by RdRp of a mitovirus in an ascomycotan ectomycorrhizal fungus. The three mitoviruses found in the three mycorrhizal fungi formed a deeply branching clade that is distinct from the two major clades in the genus Mitovirus.