The complete nucleotide sequence of an Iranian isolate of Barley yellow striate mosaic virus (BYSMV) L gene comprising 6171 nucleotides was determined using the random polymerase chain reaction followed by filling of gaps by the use of specific primers. The deduced L protein sequence of BYSMV showed similarities with the L proteins of other plant rhabdoviruses and contained polymerase module motifs characteristic of RNA‐dependent RNA polymerases of negative‐strand RNA viruses. Pairwise and multiple alignments and phylogenetic analysis of BYSMV L protein revealed that it was more closely related to cytorhabdoviruses. These results revealed that, on the basis of polymerase gene, the Iranian isolate of BYSMV and Northern cereal mosaic virus (NCMV) appeared to be the most closely related plant rhabdoviruses sequenced to date. Interestingly, the amino acid sequence identity of BYSMV/NCMV (61.3%), shared more than twice the amino acid sequence identity compared with the next two most similar cytorabdoviruses, Lettuce necrotic yellows virus (28.8%) and Lettuce yellow mottle virus (28.2%). In this paper, we discuss the similarities and differences of BYSMV with other rhabdoviruses which support the classification of BYSMV as a distinct Cytorhabdovirus. This is the first report of BYSMV genome sequences.