Flaveria (Asteraceae) is one of the few genera known to contain both C3 and C4 species, in addition to numerous biochemically-intermediate species. C3-C4 and C4-like intermediate photosynthesis have arisen more than once in different phylogenetic clades of Flaveria. Here, we characterise for the first time the photosynthetic pathway of the recently described species Flaveria kochiana B.L. Turner. We examined leaf anatomy, activity and localisation of key photosynthetic enzymes, and gas exchange characteristics and compared these trait values with those from related C4 and C4-like Flaveria species. F. kochiana has Kranz anatomy that is typical of other C4Flaveria species. As in the other C4 lineages within the Flaveria genus, the primary decarboxylating enzyme is NADP-malic enzyme. Immunolocalisation of the major C4 cycle enzymes, PEP carboxylase and pyruvate, orthophosphate dikinase, were restricted to the mesophyll, while Rubisco was largely localised to the bundle sheath. Gas exchange analysis demonstrated that F. kochiana operates a fully functional C4 pathway with little sensitivity to ambient oxygen levels. The CO2 compensation point (2.2 µbar) was typical for C4 species, and the O2-response of the CO2 compensation point was the same as the C4 species F. trinervia. Notably, F. vaginata (B.L. Robinson & Greenman), a putative C4-like species that is the nearest relative of F. kochiana, had an identical response of the CO2 compensation point to O2. Furthermore, F. vaginata, exhibited a carbon isotope ratio (–15.4‰) similar to C4 species including F. australasica Hooker, F. trinervia Spreng. C. Mohr and the newly characterised F. kochiana. F. vaginata could be considered a C4 species, but additional studies are necessary to confirm this hypothesis. In addition, our results show that F. kochiana uses an efficient C4 cycle, with the highest initial slope of the A/Ci curve of any C4Flaveria species.