Background/Question/Methods The C4 pathway has evolved independently over 50 times and its unique carbon-concentrating mechanism confers an advantage to species in high temperature, low-water environments. Differences in evolutionary lineage and biochemical subtype might be functionally important in semi-arid grasslands of North America, which often support diverse communities of C4 grass species and experience pulses in water availability. Consequently, these C4 grasses might employ different physiological drought strategies. We explored differences in photosynthetic gas exchange (net photosynthesis [Anet], stomatal conductance [gsw], instantaneous water use efficiency [WUEi]), predawn and midday water potential (Ѱp and Ѱm), stable isotopes of oxygen (δ18O) and carbon (δ13C), and morphology of five perennial grass species (Aristida purpurea var. wrightii, Bouteloua curtipendula, Erioneuron pilosum, Eriochloa sericea, Hilaria belangeri) representing three subfamilies and subtypes across three semi-arid savanna sites in Texas. We predicted that NAD-me subtype species would have the highest WUEi and maintain Anet along the aridity gradient based on previous drought-treatment studies and distribution of C4 species with mean annual precipitation. We also explored changes specific leaf area (SLA) and leaf width (Wleaf) as smaller leaves have been associated with greater WUEi and drought tolerance. Results/Conclusions We found that photosynthetic gas exchange measurements were related to both location (aridity) and species. A. wrightii and B. curtipendula sustained high levels of Anet across the aridity gradient, even with decreased gsw, likely due to comparatively higher WUEi. Shortgrasses E. pilosum and H. belangeri occupied areas with lower water availability (more negative Ѱp and enriched δ18O). These species also had the lowest Anet at the driest site and unexpectedly E. pilosum, the only NAD-me Chloridoid, had the lowest WUEi. While high conductance rates conferred a photosynthetic advantage to E. pilosum at the two wetter sites, Anet declined most dramatically for this species at the driest site. Anet and gsw remained low across the aridity gradient for E. sericea, the only Panicoid, suggesting this species exhibits a more conservative, water saving strategy. Species in the Chloridoideae family had lower δ13C, possibly due to differences in post-photosynthetic processes and/or photosynthetic efficiency. Reductions in SLA and Wleaf were not consistently observed along the aridity gradient or associated with WUEi, and may have been confounded by differences in soil N or other genus-specific environmental responses. This research highlights the importance of considering evolutionary lineage, biochemical subtype, physiognomy, and microhabitat location, all of which likely interact to produce diversity in the C4 photosynthetic response in semi-arid systems.