We analyse the isotopic values (δ 13 C, δ 15 N) of the diet of pre-Columbian horticulturalist populations from tropical and subtropical areas of southeastern South America, belonging to the Guarani and Taquara archaeological units. The data indicate different trends in each one (T = 4.21; P = 0.0004), showing a mixed diet with maize consumption in the Guarani samples (δ 13 C co = −15.5 ± 1.8‰; δ 13 C ap −10.4 ± 0.8‰) and a depleted one in the Taquara ones (δ 13 C co −18.2 ± 1.7‰; δ 13 C ap −11.9 ± 0.9‰), with a significant internal dispersion in both populations. The first population has higher nitrogen values (δ 15 N 11.1 ± 0.6‰) compared to the Taquara samples (δ 15 N 9.3 ± 1‰), suggesting a more carnivorous diet. The recognition of these pre-Columbian mixed diets involves the identification of maize cultivation on the Atlantic side of the southernmost area of South America (Parana Delta, 34°SL). Through the analysis of δ 18 O we have identified two isotopic ecozones, the first along the Paraná River Valley, with an average value of δ 18 O −3.7 ± 0.5‰ (CV = 13.5%; CI = −3.83 / −3.16), and the second one, located in the Planalto of southern Brazil (Araucaria Forest), with a mean value of δ 18 O −1.5 ± 0.3‰ (CV = 16.5%; CI = −1.69 / −1.29). The isotopic data (δ 13 C, δ 15 N and δ 18 O) suggest human movements between these two ecozones.