Abstract. The dissolved humic substances of seven Venezuelan blackwater rivers, belonging to different tropical biogeographical regions, were analyzed. Fractionation using Sephadex gel together with an analysis of their fluorescence, absorbance and infrared spectral properties, showed differences between their humic substances. These differences were related to their biological sources and humification environment. Statistical analysis of the properties of these humic substances allowed us to separate the rivers into three groups. These groups were directly related to the geological features and vegetation of the terrestrial ecosystems into which they drain: -The Autana, Atabapo, Chola, Negro which belong to the Amazon basin on the Guayana Shield, each with typical blackwater riverine forest. -The Caroni, belonging to the Orinoco basin on the Guayana Shield and with tropical humid forest and rainforest. -The Morichal Largo which draines the Mesa Formation from the eastern Llanos and has gallery forest of MauririafZexuosa.