ABSTRACT: BACKGROUND:The basic helix-loop-helix transcription factors play important roles in diverse cellular and molecular processes. Comparative functional genomics can provide powerful approaches to draw inferences about gene function and evolution among species. The comprehensive comparison of bHLH gene family in different gramineous plants has not yet been reported. RESULTS:In this study, a total of 183, 231 and 571 bHLHs were identified in rice, maize and wheat genomes respectively, and 1154 bHLH genes from the three species and Arabidopsis were classified into 36 subfamilies. Of the identified genes, 110 OsbHLHs, 188 ZmbHLHs and 209 TabHLHs with relatively high mRNA abundances were detected in one or more tissues during development, and some of them exhibited tissue-specific expression such as TabHLH454-459, ZmbHLH099-101 and OsbHLH037 in root, TabHLH559-562, -?046, -?047 and ZmbHLH010, -?072, -?226 in leaf, TabHLH216-221, -?333, -?335, -?340 and OsbHLH005, -?141 in inflorescence, TabHLH081, ZmbHLH139 and OsbHLH144 in seed. Forty five, twenty nine and thirty one differentially expressed bHLHs were respectively detected in wheat, maize and rice under drought stresses using RNA-seq technology. Among them, the expressions of TabHLH046, -?047, ZmbHLH097, -?098, OsbHLH006 and?-?185 were strongly induced, whereas TabHLH303, -?562, ZmbHLH155, -?154, OsbHLH152 and?-?113 showed significant down-regulation. Twenty two TabHLHs were induced after stripe rust infection at 24?h and nine of them were suppressed at 72 hpi, whereas 28 and 6 TabHLHs exhibited obviously down- and up-regulation after powdery mildew attack respectively. Forty one ZmbHLHs were differentially expressed in response to F. verticillioides infection. Twenty two co-expression modules were identified by the WGCNA, some of which were associated with particular tissue types. And GO enrichment analysis for the modules showed that some TabHLHs were involved in the control of several biological processes, such as tapetal PCD, lipid metabolism, iron absorption, stress responses and signal regulation. CONCLUSION:The present study identifies the bHLH family in rice, maize and wheat genomes, and detailedly discusses the evolutionary relationships, expression and function of bHLHs. This study provides some novel and detail information about bHLHs, and may facilitate understanding the molecular basis of the plant growth, development and stress physiology.