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This study systematically evaluates the presence of methane mitigating metabolites in two hemp (<i>Cannabis sativa</i> L.) varieties, <i>Futura 75</i> and <i>Finola</i>. Hemp metabolites were extracted with methanol and fractionated using Solid Phase Extraction (SPE). Extracts, fractions, and the remaining pulp were screened for their methane mitigating potential using an in vitro model of rumen fermentation. The bioactive metabolites were identified with Liquid Chromatography-Mass Spectrometry (LC-MS). When incubated with a standard feed (maize silage), the extract of <i>Futura 75</i> significantly reduced methane production compared to that of control (without added extract) and without negative effects on feed degradability and volatile fatty acid patterns. The compounds responsible for the methane mitigating effect were assigned to flavonoid glycosides. However, none of the fractions of <i>Futura 75</i> or the pulp exhibited similar effect on methane emission. Butyric acid concentration in the fermentation inoculum was significantly increased, which could indicate why methane production was higher, when incubated with the fractions and the pulp. The extract of <i>Finola</i> did not show a similar significant effect, however, there was a numerical tendency towards lower methane production. The difference in methane mitigating properties between <i>Cannabis sativa</i> L. <i>Futura 75</i> and <i>Finola</i>, could be related to the content of bioactive flavonoids.