The BES1 (BRI1-EMSSUPPRESSOR1) gene family is a unique class of transcription factors that play dynamic roles in the Brassinosteroids (BRs) signaling pathway. An approach of genome-sequencing in a large number of plants was employed, which permitted the molecular characterization and a comprehensive functional study on the BES1 gene family for their potential roles in the development of agronomic traits and stress responses. A total of 135 BES1 genes were recognized and characterized, which were divided into 5 well-conserved subfamilies, consistent with the topology with statistical support and the profound duplication knots of BES1 paralogues. BES1 was not found in lower plants as represented by glaucophyta, rhodophyte and chlorophyta. The spatial expression profiles of BES1s in Arabidopsis, rice and cotton, as well as their response to abiotic stresses were analyzed. Overexpression of two rice BES1 genes, i.e OsBES1-3 and OsBES1-5, promotes root growth under drought stress. Overexpression of GhBES1-4 from cotton enhanced salt tolerance in Arabidopsis. Five protein interaction networks were constructed and numerous genes co-expressed with GhBES1-4 were characterized in transgenic Arabidopsis. BES1 may have evolved in the ancestor of first land plants following its divergence from algae. Overexpression of BES1 gene improved the stress tolerance of plants.