A variety of methods for the detection and characterization of fungal viruses are available. For many years, serological and biological assays were used for virus detection. Today, more sensitive methods like polymerase chain reaction, together with sequencing, are widely used to study viruses. Extracting double-stranded (ds) RNA can be a useful approach to detect and study mycoviruses from fungal tissues, as dsRNAs accumulate in infected cells as copies of viral genomes or as replicative intermediates of single-stranded RNA genomes. Here we present a basic protocol for growing fungal strains and isolating dsRNA using cellulose chromatography, followed by molecular diagnostic methods including cDNA synthesis, sequencing, and determination of 5′ ends by primer ligation.