Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, causes serious crop losses in agriculture. Here, we have cloned the BYSMV-derived small interfering RNAs (siRNAs), assembled the siRNAs and used RT-PCR to reconstruct the BYSMV genome. The genome consists of 12,706 nucleotides and encodes ten predicted genes from the antigenomic strand. The major BYSMV structural proteins share identities ranging from 35% to 62% with northern cereal mosaic virus (NCMV) counterparts. A notable difference is that BYSMV contains three transcriptional units residing between the P and M genes compared with four units in the corresponding region of NCMV. Unexpectedly, the middle mRNA in this region encodes gene5 nested in an alternative frame within gene4 via a leaky scanning mechanism. The gene5 encodes a small hydrophobic protein targeting to the endoplasmic reticulum (ER). To our knowledge, this is the first report of nested gene in plant rhabdoviruses.