In vitro methane production was determined in mixtures of grasses and legumes of Colombian highlands were evaluated using the gas production technique. Samples of two grasses, ryegrass (Lolium perenne) and kikuyu (Pennisetum clan-destinum), and two legumes, white clover (Trifolium repens) and big trefoil (Lotus uli-ginosus), from two different paddocks were harvested. The treatments were mixtures of kikuyu and lotus, kikuyu and white clover, ryegrass and lotus and ryegrass and clover in three different grass: legumes ratio (90:10, 70:30 and 50:50). The methane concentration in the gas was determined by gas chromatography. Methane production per unit of degraded dry matter decreased 14 % (p<0.05) when the proportions of legume increased associated with kikuyu. However, no differences were observed when legumes were mixed with ryegrass. When proportion of lotus, a tanniferous legume, increased total methane production decreased independently of grass suggesting an effect in methanogenic population.