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 C₄ Flaveria 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/C_i curve of any C₄ Flaveria species.