Summary: Our study investigated the effects of condensed tannins (CT) on rumen in vitro methane (CH₄) production and fermentation characteristics by incubating lucerne in buffered rumen fluid in combination with different CT extracts at 0 (control), 40, 80 and 120 g CT/kg of substrate DM. Condensed tannins were extracted from four sainfoin accessions: Rees “A”, CPI63763, Cotswold Common and CPI63767. Gas production (GP) was measured using a fully automated GP apparatus with CH₄ measured at distinct time points. Condensed tannins differed substantially in terms of polymer size and varied from 13 (Rees “A”) to 73 (CPI63767) mean degree of polymerization, but had relatively similar characteristics in terms of CT content, procyanidin:prodelphinidin (PC:PD) and cis:trans ratios. Compared to control, addition of CT from CPI63767 and CPI63763 at 80 and 120 g CT/kg of substrate DM reduced CH₄ by 43% and 65%, and by 23% and 57%, respectively, after 24 h incubation. Similarly, CT from Rees “A” and Cotswold Common reduced CH4 by 26% and 46%, and by 28% and 46%, respectively. Addition of increasing level of CT linearly reduced the maximum rates of GP and CH4 production, and the estimated in vitro organic matter digestibility. There was a negative linear and quadratic (p < 0.01) relation between CT concentration and total volatile fatty acid (VFA) production. Inclusion of 80 and 120 g CT/kg DM reduced (p < 0.001) branched chain VFA production and acetate:propionate ratio and was lowest for CPI63767. A decrease in proteolytic activity as indirectly shown by a change in VFA composition favoring a shift towards propionate and reduction in branched-chain VFA production varied with type of CT, and was highest for CPI63767. In conclusion, these results suggest that tannin polymer size is an important factor affecting in vitro CH₄ production which may be linked to the CT interaction with dietary substrate or microbial cells.