Several mud volcanoes are active in the Canadian Beaufort Sea. In this study, we investigated vertical variations in methanotrophic communities in sediments of the mud volcano MV420 (420 m water depth) by analyzing geochemical properties, microbial lipids, and nucleic acid signatures. Three push cores were collected with a remotely operated vehicle from visually discriminative habitats that were devoid of megafauna and/microbial mats (DM) to the naked eye, covered with bacterial mats (BM), or colonized by siboglinid tubeworms (ST). All MV420 sites showed the presence of aerobic methane oxidation (MOx)- and anaerobic methane oxidation (AOM)-related lipid biomarkers (4α-methyl sterols and sn-2-hydroxyarchaeol, respectively), which were distinctly different in comparison with a reference site at which these compounds were not detected. Lipid biomarker results were in close agreement with 16S rRNA analyses, which revealed the presence of MOx-related bacteria (Methylococcales) and AOM-related archaea (ANME-2 and ANME-3) at the MV420 sites. 4α-methyl sterols derived from Methylococcales predominated in the surface layer at the BM site, which showed a moderate methane flux (0.04 mmol cm-2 y-1), while their occurrence was limited at the DM (0.06 mmol cm-2 y-1) and ST (0.01 mmol cm-2 y-1) sites. On the other hand, 13C-depleted sn-2-hydroxyarchaeol potentially derived from ANME-2 and/or ANME-3 was abundant in down-core sediments at the ST site. Our study indicates that a niche diversification within this mud volcano system has shaped distinct methanotrophic communities due to availability of electron acceptors in association with varying degrees of methane flux and bioirrigation activity.