Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes. A major step in control of viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. We used barley yellow dwarf virus (BYDV) as a model system to study transcriptional control in a virus with multiple sgRNAs. BYDV generates three sgRNAs during infection. The sgRNA1 promoter has been mapped previously to a 98-nucleotide (nt) region which forms two stem-loop structures. It was determined that sgRNA1 is not required for BYDV RNA replication in oat protoplasts. In this study, we show that neither sgRNA2 nor sgRNA3 is required for BYDV RNA replication. The promoters for sgRNA2 and sgRNA3 synthesis were mapped by using deletion mutagenesis. The minimal sgRNA2 promoter is approximately 143 nt long (nt 4810 to 4952) and is located immediately downstream of the putative sgRNA2 start site (nt 4809). The minimal sgRNA3 core promoter is 44 nt long (nt 5345 to 5388), with most of the sequence located downstream of sgRNA3 start site (nt 5348). For both promoters, additional sequences upstream of the start site enhanced sgRNA promoter activity. These promoters contrast to the sgRNA1 promoter, in which almost all of the promoter is located upstream of the transcription initiation site. Comparison of RNA sequences and computer-predicted secondary structures revealed little or no homology between the three sgRNA promoter elements. Thus, a small RNA virus with multiple sgRNAs can have very different subgenomic promoters, which implies a complex system for promoter recognition and regulation of subgenomic RNA synthesis.