Methane (CH4) is usually present in trace quantities at the sediment-water interface in riverine environments. Unusually, along the Condamine River in Queensland, several CH4 macro-seeps occur which can elevate water-column concentrations up to three orders of magnitude above levels recorded in unaffected river reaches. Anecdotal reports also indicate that these CH4 macro-seeps can form and diminish over annual to decadal timeframes. Aerobic methanotrophs metabolise CH4 (via CH4 oxidation) as their only source of carbon and energy, and can remove CH4 from the water column before evasion to the atmosphere. However, the degree to which methanotroph communities metabolise CH4, and also how rapidly they respond to new CH4 macro-seeps, is still unknown in the Condamine River. In this study, we (a) quantified rates of CH4 oxidation at the sediment-surface interface in macro-seep and unaffected river reaches, and (b) performed a sediment translocation experiment to investigate how quickly benthic methanotroph communities respond to the formation of new CH4 macro-seeps. Rates of CH4 oxidation were, on average, between 30 and 235 times greater in the vicinity of CH4 macro-seeps compared to unaffected reaches. Rates of CH4 oxidation of translocated sediment increased rapidly when exposed to CH4 macro-seeps and approached rates quantified at macro-seep reaches within 7 days. Periods of high-flow appeared to suppress rates of CH4 oxidation. Our results indicate that, under aerobic conditions, benthic methanotroph communities can rapidly develop and take advantage of this abundant and new carbon source if new macro-seeps develop. Ongoing work is quantifying changes in benthic microbial community composition to the presence of new CH4 macro-seeps and if CH4–derived carbon constitutes a major source of energy for local aquatic food webs.