C₄ photosynthesis has evolved many times in 18 different families of land plants with great variation in leaf anatomy, ranging from various forms of Kranz anatomy to C₄ photosynthesis occurring within a single type of photosynthetic cell. There has been little research on photosynthetic typing in the family Cleomaceae, in which only one C₄ species has been identified, Cleome gynandra L. There is recent interest in selecting and developing a C₄ species from the family Cleomaceae as a model C₄ system, since it is the most closely related to Arabidopsis, a C₃ model system (Brown et al. 2005). From screening more than 230 samples of Cleomaceae species, based on a measure of the carbon isotope composition (δ¹³C) in leaves, we have identified two additional C₄ species, C. angustifolia Forssk. (Africa) and C. oxalidea F.Muell. (Australia). Several other species have δ¹³C values around –17‰ to –19‰, suggesting they are C₄-like or intermediate species. Eight species of Cleome were selected for physiological, anatomical and biochemical analyses. These included C. gynandra, a NAD–malic enzyme (NAD–ME) type C₄ species, C. paradoxa R.Br., a C₃–C₄ intermediate species, and 6 others which were characterised as C₃ species. Cleome gynandra has C₄ features based on low CO₂ compensation point (Γ), C₄ type δ¹³C values, Kranz-type leaf anatomy and bundle sheath (BS) ultrastructure, presence of C₄ pathway enzymes, and selective immunolocalisation of Rubisco and phosphoenolpyruvate carboxylase. Cleome paradoxa was identified as a C₃–C₄ intermediate based on its intermediate Γ (27.5 μmol mol^–1), ultrastructural features and selective localisation of glycine decarboxylase of the photorespiratory pathway in mitochondria of BS cells. The other six species are C₃ plants based on Γ, δ¹³C values, non-Kranz leaf anatomy, and levels of C₄ pathway enzymes (very low or absent) typical of C₃ plants. The results indicate that this is an interesting family for studying the genetic basis for C₄ photosynthesis and its evolution from C₃ species.