Shelf sea areas are the primary oceanic source for methane release, the most abundant hydrocarbon in the atmosphere. As such, the southern North Sea’s methane concentration is mainly determined by river runoff and tidal marshes. Within such a highly variable temperate estuary, this study is the first to reveal detailed information on the in situ activity, abundance and community structure of methane oxidizing bacteria along a transect from the marine environment near Helgoland island to the riverine harbor of Hamburg, Germany. The in situ methane oxidation rate was determined with a radio tracer, and methane concentration with the head-space method. Abundance and diversity of the methanotrophic bacterial community in the water column was assessed with quantitative polymerase chain reaction for the particulate methane monooxygenase and monooxygenase intergenic spacer analysis. Median abundances ranged from 2.8 × 104 cells l−1 in the marine environment to 7.5 × 105 cells l−1 in the riverine environment. Except for salinity, no conclusive linear correlation between any environmental parameter and the abundance of methanotrophs could be determined. Relating activity with abundance of methanotrophs showed that about 70% of the population accounted for is inactive, especially in the coastal and marine environment. This study found distinct operational taxonomic unit (OTU) community compositions among the 3 environmental categories (river, coast, marine). Several identified OTUs have been reported previously and imply a wide geographic occurrence. Overall, we propose that salinity is the most important driver in shaping differing methanotrophic communities in the riverine, coastal and marine environment.