The infrared radiofluorescence (IR-RF) dating technique was applied to eight fluvial samples that were collected from two sediment cores at the Heidelberg Basin located near Viernheim and Ludwigshafen in southwest Germany. Based on the IR-RF derived ages of the samples it was possible to establish a chronological framework for the Mid-Pleistocene fluvial deposits of the Heidelberg Basin. The results allow us to distinguish between four main periods of aggradation. The lowermost sample taken from 100 m core depth lead to an IR-RF age of 643 ± 28 ka pointing to a Cromerian period of aggradation (OIS 17–16). For the Elsterian it is now possible to distinguish between two aggradation periods, one occurring during the Lower Elsterian period (OIS 15) and a second during the Upper Elsterian period (OIS 12–11). For the so called Upper interlayer (or “Oberer Zwischenhorizont” — a layer of organic-rich and finer-grained deposits), the IR-RF results point to a deposition age of around 300 ka, with samples taken directly on top and out of this layer yielding IR-RF ages of 288 ± 19 ka and 302 ± 19 ka, respectively. Hence, the measured IR-RF ages clearly point to a deposition during the Lower Saalian period (OIS 9–8) whereas earlier studies assumed a Cromerian age for the sediments of the Upper Interlayer based on pollen records and also mollusc fauna. The new IR-RF dataset indicates that significant hiatuses are present within the fluvial sediment successions. In particular the Eemian and Upper Saalian deposits are missing in this part of the northern Upper Rhine Graben, as the 300 ka deposits are directly overlain by Weichselian fluvial sediments. It is obvious that time periods of increased fluvial aggradation were interrupted by time periods of almost no aggradation or erosion which should have been mainly triggered by phases of increased and decreased subsidence of the Heidelberg Basin.