Methane is an important greenhouse gas and the Elbe River carries high amounts of methane into the North Sea. Methane oxidizing bacteria do have the capability to suppress the release of methane into the atmosphere. However, little is known about the diversity and community composition of these bacteria in an estuarine water column. To gain insight in that matter, the following study extended the field of application of the fingerprinting method monooxygenase intergenic spacer analysis (MISA), from a so far marine to a more complex estuarine environment. For separation of the obtained fingerprints two electrophoretic separation systems, the DNA Analyzer 4300 (Licor) and the Multi NA (Shimadzu) were compared and the better suited system was used to analyze environmental samples along a transect from Hamburg to Helgoland. Sampling took place every two months from November 2013 to November 2014. Besides the analysis of diversity and community structure of methanotrophic bacteria, potential environmental driving forces were determined and limitations of the applied MISA method were discussed. Experiments of this study showed that the DNA Analyzer 4300 has a higher resolution power with respect to the obtained DNA fragments of the MISA fingerprinting method. In addition as compared to the Multi NA the DNA Analyzer 4300 revealed a better reproducibility of results. MISA results of the environmental samples reveal changes in community composition and diversity on a temporal and spatial scale. Due to the salinity gradient along the Hamburg- Helgoland transect, three environmental zones were determined, of which the coastal zone showed the highest diversity estimates. Principal coordinate analysis (PCoA) revealed a shift in community composition between the riverine and coastal/marine zone. Distance based redundancy analysis (dbRDA) of environmental variables determined “salinity” as the main driver of the observed changes in methanotrophic community composition along the Hamburg-Helgoland transect.