Carbon sequestration has become an important issue in forest management in the light of concerns about global warming. The two methods proposed by IPCC to quantify the net carbon sequestered by trees are based either on the estimation of annual gains and losses of carbon due to the growth of trees and their removals (default method), or on the difference in carbon stocks between two consecutive inventories (stock-change method). The objectives of this study are to compare the net carbon sequestered by the tree layer of forest ecosystems in Andalusia estimated by the two methods, and to identify what measured variables contribute the most to the differences in the estimations. Multivariate analysis was performed in order to identify the causes of these variations. The results revealed differences of up to 200% in the estimated net carbon sequestration depending on the methodology employed, and changes in the direction of the capture/emission in some areas. The net carbon sequestration ranged between 0.08 ± 0.01 and 1.93 ± 0.52 Mg C ha−1 year−1 depending on the method used and on the tree species analyzed. In global terms, net carbon sequestration at the regional level reaches 2,101,715 ± 560,906 Mg C according to the default method and 1,693,412 ± 272,529 Mg C using the stock-change method. © 2017 Informa UK Limited, trading as Taylor & Francis Group.