BackgroundAbscisic acid (ABA) receptor pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptor proteins (PYR/PYL/RCARs) have been demonstrated to play pivotal roles in ABA signaling and in response to diverse environmental stimuli including drought, salinity and osmotic stress inArabidopsis. However, whether and how GhPYL9-5D and GhPYR1-3A, the homologues ofArabidopsisPYL9 and PYR1 in cotton, function in responding to ABA and abiotic stresses are still unclear.ResultsGhPYL9-5D and GhPYR1-3A were targeted to the cytoplasm and nucleus. Overexpression ofGhPYL9-5DandGhPYR1-3AinArabidopsiswild type and sextuple mutantpyr1pyl1pyl2pyl4pyl5pyl8plants resulted in ABA hypersensitivity in terms of seed germination, root growth and stomatal closure, as well as seedling tolerance to water deficit, salt and osmotic stress. Moreover, the VIGS (Virus-induced gene silencing) cotton plants, in whichGhPYL9-5DorGhPYR1-3Awere knocked down, showed clearly reduced tolerance to polyethylene glycol 6000 (PEG)-induced drought, salinity and osmotic stresses compared with the controls. Additionally, transcriptomic data revealed thatGhPYL9-5Dwas highly expressed in the root, andGhPYR1-3Awas strongly expressed in the fiber and stem.GhPYL9-5D,GhPYR1-3Aand their homologs in cotton were highly expressed after treatment with PEG or NaCl, and the two genes were co-expressed with redox signaling components, transcription factors and auxin signal components. These results suggest that GhPYL9-5D and GhPYR1-3A may serve important roles through interplaying with hormone and other signaling components in cotton adaptation to salt or osmotic stress.ConclusionsGhPYL9-5D and GhPYR1-3A positively regulate ABA-mediated seed germination, primary root growth and stomatal closure, as well as tolerance to drought, salt and osmotic stresses likely through affecting the expression of multiple downstream stress-associated genes inArabidopsisand cotton.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04330-8.