This study analyses the regressive phase of the marine Froidefontaine Subgroup and the subsequent fluviolacustrine Niederroedern formation in the southern Upper Rhine Graben during the Late Rupelian and the position of the Rupelian/Chattian boundary. The study is based on the sedimentary record and several microfossil groups from two boreholes, with a focus on new records of fish otoliths, Bolboforma and Charophyta. The biostratigraphic evaluation of these groups provides evidence for a position of the Rupelian/Chattian boundary within the upper Niederroedern Formation. This is contrary to the results from a previous sedimentological approach, which places the boundary at the base of the Niederroedern Formation. The fish fauna indicates a biogeographic relation to southern France, the Molasse Basin of Switzerland and southern Germany.