Archaeobotanical remains of Setaria grains and chaff were found at the Chengtoushan site in south-central China (ca. 5800 cal b.p.). Grain shape was determined, using length to breadth ratios, and morphological variation in the upper lemma of modern domesticated and wild Setaria species were examined using scanning electron microscopy as a basis for identifying archaeobotanical remains. Grains of S. viridis, S. yunnanensis, and S.×pycnocoma are slender, whereas S. italica, S. italica var. germinica, S. lutescenes, S. faberi, S. glauca, S. pallidefusca and S. intermedia are round in shape. The papillae distributed on the upper lemma of S. italica are small (8–15 μm) with a non-ridged base, while other Setaria species have large papillae (15–20 μm) with a widely ridged base. The remains of the Setaria from the Neolithic layers at Chengtoushan included S. italica, based on these identification characters. These new finds of foxtail millet are the earliest discoveries from the Yangtze River basin of southern China and are also the earliest evidence for co-cultivation of foxtail millet with rice. The implications of these findings for understanding foxtail millet domestication centres are discussed.