Cleomaceae is one of 19 angiosperm families in which C(4) photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C(4) in genus Cleome. Methods Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC).Three species with C(4)-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C(4) photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C(4) cycle (compared with the C(3)-C(4) intermediate C. paradoxa and the C(3) species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells.NAD-malic enzyme-type C(4) photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C(3)-C(4) intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.