The forage legume species biserrula (Biserrula pelecinus) is among a few forage species with low enteric methane emission when fermented by rumen microbes and it is unclear whether metabolites in biserrula play a role in this. The hypothesis of this study was that specific metabolites in biserrula are candidate biomarkers for and associated with low methanogenesis. We characterized the metabolomic profiles of 30 accessions of biserrula or its core collection using nuclear magnetic resonance and liquid chromatography mass spectrometry. These profiles were then analyzed for association with methanogenesis potential in the rumen. Metabolomic profile was predictive of methanogenesis. Forty-seven putative metabolites were identified by liquid chromatography mass spectrometry that were highly expressed (P < 0.05) in the associations with low methanogenic potential. Fragmentation analysis of a subset of these metabolites suggested saponins, with one feature tentatively identified as an ursolic or oleanolic terpene glucoside, consistent with the nuclear magnetic resonance data. Accessions with Eritrean geographic origin were metabolomic outliers. Here, we show for the first time that some metabolites contribute to the methane mitigation effects of biserrula. If in vivo results confirm this, the environmental impact of this study would be the availability of biomolecules for livestock vaccination to mitigate methane emission resulting in an economic impact of lower cost of production in countries with a price on environmental emissions.