The Atlantic Forest, Humid Chaco, and Dry Chaco are major eco-regions in Paraguay, but information on forest carbon stocks in these forest types remains limited. To establish a system to measure, report and verify forest carbon change under the REDD+ mechanism, we developed new allometric equations to estimate tree biomass in each eco-region. Three models of total and aboveground biomass were developed from destructive sampling data. The models performed well, explaining ≥96% of the variation in aboveground and total biomass, although the best model differed among the eco-regions. The inclusion of height and wood density improved fit in the Atlantic Forest model, but including wood density did not enhance the Humid and Dry Chaco models. Our models will improve the estimation of biomass in Paraguay because they provide better estimates of total and aboveground biomass in each eco-region than pan-tropical generic models.