A new double-stranded (ds) RNA mycovirus has been identified in isolate Ds752-1 of the phytopathogenic fungus Dothistroma septosporum, the causal agent of Dothistroma needle blight, also known as red band needle blight or pine needle blight. Dothistroma septosporum chrysovirus 1 (DsCV-1) is a new member of the genus Alphachrysovirus in the family Chrysoviridae. The DsCV-1 genome comprises four dsRNA elements designated 1, 2, 3, and 4 from largest to smallest. dsRNA1 encodes an RNA-dependent RNA polymerase (RdRP) that is most similar to the RdRP of Erysiphe necator associated chrysovirus 3. dsRNA2 potentially encodes two hypothetical proteins, one of which is small and has no homology to known proteins, and one of which is large with significant sequence similarity to the alphachryso-P3 of other alphachrysoviruses. dsRNA3 and dsRNA4 encode a coat protein (CP) and a putative cysteine protease, respectively. This is the first report of a mycovirus infecting the fungus D. septosporum, and DsCV-1 is one of three Chrysoviridae family members found to possess genomic dsRNAs potentially encoding more than one protein.