Barley is an economically important crop widely cultivated in the world. Boron (B) toxicity limits its yield in a variety of regions worldwide. B-tolerant cultivar of barley (Hordeum vulgare, cv Sahara) maintains low B in their roots and leaves in B-toxic conditions and able to resist against it. B import and export to the plant is controlled by several transmembrane proteins such as aquaporins (AQP), which are key players not just for water uptake, but also a wide selection of substrates including B. In this study, we identified and characterized AQPs from B-tolerant barley to understand their possible positive impact in B-toxicity tolerance. In order to filter out the AQP genes, we have mined the RNA-Seq data obtained from barley roots and leaves which are B-stressed and control in this study. A total of 30 AQP were identified within four subfamilies: plasma membrane intrinsic proteins (PIPs) (13), tonoplast intrinsic proteins (TIPs) (11), NOD26-like intrinsic proteins (NIPs) (4), and small basic intrinsic proteins (SIPs) (2). Differential expression was measured in barley AQP transcripts upon excess B treatment. Contribution of each AQP member to B-tolerance was evaluated. Particularly, NIP1:1 was found to be highly up-regulated in roots, in contrast down-regulated in leaf, indicating plasma membrane and vacuole cooperated control of B-regulation. In addition to NIP2:1, NIP2:2 expression was found to be reduced that may contribute to B-toxicity tolerance. Moreover, comparative phylogenetic analysis was conducted using diverse plant AQP members. Comprehensive evaluation of conserved domains, critical residues, and 3D models give insights about the substrate selectivity and passage capability of barley AQPs.