C(4) photosynthesis evolved multiple times in the Amaranthaceae s.s., but the C(4) evolutionary lineages are unclear because the photosynthetic pathway is unknown for most species of the family. To clarify the distribution of C(4) photosynthesis in the Amaranthaceae, we determined carbon isotope ratios of 607 species and mapped these onto a phylogeny determined from matK/trnK sequences. Approximately 28% of the Amaranthaceae species use the C(4) pathway. C(4) species occur in 10 genera-Aerva, Amaranthus, Blutaparon, Alternanthera, Froelichia, Lithophila, Guilleminea, Gomphrena, Gossypianthus, and Tidestromia. Aerva, Alternanthera, and Gomphrena contain both C(3) and C(4) species. In Aerva, 25% of the sampled species are C(4). In Alternanthera, 19.5% are C(4), while 89% of the Gomphrena species are C(4). Integration of isotope and matK/trnK data indicated C(4) photosynthesis evolved five times in the Amaranthaceae, specifically in Aerva, Alternanthera, Amaranthus, Tidestromia, and a lineage containing Froelichia, Blutaparon, Guilleminea, Gomphrena pro parte, and Lithophila. Aerva and Gomphrena are both polyphyletic with C(3) and C(4) species belonging to distinct clades. Alternanthera appears to be monophyletic with C(4) photosynthesis originating in a terminal sublineage of procumbent herbs. Alpine C(4) species were also identified in Alternanthera, Amaranthus, and Gomphrena, including one species (Gomphrena meyeniana) from 4600 m a.s.l.