This study aimed to develop a biomass equation for estimating the total above-ground biomass for Colophospermum mopane (mopane) based on the pooled data from three study sites. The mopane woodlands in Botswana represent 14.6% of Botswana's total area. The woodlands directly or indirectly support the livelihood of the majority of the rural population by providing wood and non-wood products. However, there is limited information on the pattern, trends and distribution of woody biomass production and their primary, environmental, and climatic determinants in different parts of Botswana. All the data were collected by destructive sampling from three study sites in Botswana. Stratified random sampling was based on the stem diameter at breast height (1.3 m from the ground or Diameter at Breast Height (DBH)). A total of 30 sample trees at each study site were measured, felled and weighed. The data from the three sites were pooled together, and the study employed regression analysis to examine the nature of relationships between total above-ground biomass (dependent variable) and five independent variables: 1) total tree height; 2) crown diameter; 3) stem diameters at 0.15 m; 1.3 m (DBH) and 3 m from the ground respectively. There were significant relationships between all the independent variables and the dependent variable. However, DBH emerged as the strongest predictor of total tree above-ground biomass for mopane. The equation ln 1.163 2.190 ln Biomass DBH = − + was adopted for use in the indirect estimation of total tree above-ground biomass for mopane in Botswana.