Stevia rebaudianaBertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs.bHLHtranscription factors play a crucial role in plant development and secondary metabolism. In this study, 159SrbHLHgenes were identified from theS. rebaudianagenome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events ofSrbHLHgenes were also studied. Moreover, based on the RNA-Seq data of different tissues ofS. rebaudiana, 28SrbHLHswere co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidateSrbHLHgenes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function ofSrbHLHsin regulating SGs biosynthesis and lays the foundation for future applications ofSrbHLHgenes in molecular breeding ofS. rebaudiana.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04353-1.