Environmental change requires more crop production per water use to meet the rising global food demands. However, improving crop intrinsic water use efficiency (iWUE) usually comes at the expense of carbon assimilation. Sorghum is a key crop in many vulnerable agricultural systems with higher tolerance to water stress (WS) than most widely planted crops. To investigate physiological controls on iWUE and its inheritance in sorghum we screened 89 genotypes selected based on inherited haplotypes from an elite or five exotics lines, containing a mix of geographical origins and dry vs. milder climates, which included different aquaporin (AQP) alleles. We found significant variation among key highly heritable gas exchange and hydraulic traits, with some being significantly affected by variation in haplotypes among parental lines. Plants with a higher proportion of the non-stomatal component of iWUE still maintained iWUE under WS by maintaining photosynthetic capacity, independently of reduction in leaf hydraulic conductance. Haplotypes associated with two AQPs (SbPIP1.1 and SbTIP3.2) influenced iWUE and related traits. These findings expand the range of traits that bridge the trade-off between iWUE and productivity in C4 crops, and provide possible genetic regions that can be targeted for breeding.