This paper presents the results of the environmental characterization of the stillage from the production of second generation ethanol from two sources: the first is the synthetic substrate produced in the laboratory to simulate the conditions of cane waste, and the second comes hydrolyzed of sugar cane waste. This is to identify treatment alternatives to reduce their impact and its contamination potential. In the characterization of stillage were determined important parameters for the quality of them, such as Biochemical Oxygen Demand (BOD 5 ), Chemical Oxygen Demand (COD), suspended solids, nitrates, ammonia, phosphates, etc. It was found that the stillage have a lower pollutant load when it is compared to other stillage from a process with a different raw material. However, they have high BOD 5 and COD values, as well as, nitrates, phosphates and total solids which exceed the Colombian law for effluent discharges.