Engineering C 4 photosynthesis into C 3 crops such as rice or wheat could substantially increase their yield by alleviating photorespiratory losses 1,2 . This objective is challenging because the C 4 pathway involves complex modifications to the biochemistry, cell biology and anatomy of leaves 3 . Forward genetics has provided limited insight into the mechanistic basis of these characteristics and there have been no reports of significant quantitative intra-specific variation of C 4 attributes that would allow trait-mapping 4,5 . Here we show that accessions of C 4 Gynandropsis gynandra collected from locations across Africa and Asia exhibit natural variation in key characteristics of C 4 photosynthesis. Variable traits include bundle sheath size and vein density, gas exchange parameters and carbon-isotope discrimination associated with the C 4 state, but also abundance of transcripts encoding core enzymes of the C 4 cycle. Traits relating to water use showed more quantitative variation than those associated with carbon assimilation. We propose variation in these traits likely adapted the hydraulic system for increased water use efficiency rather than improving carbon fixation, indicating that selection pressure may drive C 4 diversity in G. gynandra by acting to modify water use rather than photosynthesis. As these accessions can be easily crossed and produce fertile offspring, our findings indicate that natural variation within a C 4 species is sufficiently large to allow genetic-mapping of key anatomical C 4 traits and regulators.