Biserrula (Biserrula pelecinus L.) is an important annual pasture legume for the wheatbelt of southern Australia and has been found to have lower levels of methane output than other pasture legumes when fermented by rumen microbes. Thirty accessions of the biserrula core germplasm collection were grown in the glasshouse to examine intra-specific variability in in vitro rumen fermentation, including methane output. One biserrula cultivar (Casbah) was also grown at two field locations to confirm that low methanogenic potential was present in field-grown samples. All of the biserrula accessions had significantly reduced methane [range 0.5–8.4 mL/g dry matter (DM)] output compared with subterranean clover (28.4 mL/g DM) and red clover (36.1 mL/g DM). There was also significant variation in fermentability profiles (except for volatile fatty acids) among accessions of the core collection. Methanogenic potential exhibited 86% broad-sense heritability within the biserrula core collection. The anti-methanogenic and gas-suppressing effect of biserrula was also confirmed in samples grown in the field. In conclusion, biserrula showed variability in in vitro fermentation traits including reduced methane production compared with controls. This bioactivity of biserrula also persists in the field, indicating scope for further selection of biserrula as an elite methane-mitigating pasture.