Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C4 acid decarboxylases, and immunolocalization of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and the P-subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C3 plants, while Heliotropium texanum and Heliotropium polyphyllum are C4 species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C3, but exhibit ‘proto-Kranz’ anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C3–C4 intermediates, with Kranz-like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C3–C4 species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C4 evolution. Where existing models such as Flaveria emphasize diversification of C3–C4 intermediates, Heliotropium has numerous C3 species expressing proto-Kranz traits that could represent a critical initial phase in the evolutionary origin of C4 photosynthesis.