Locally extinct since the 1960s, the anadromous allis shad (Alosa alosa) was reintroduced into the Rhine system through a restocking programme beginning in 2007. The population is now showing positive signs of recovery, with natural reproduction occurring for several years and a decreasing proportion of stocked fish. These findings suggest the future establishment of a self-sustaining population. Our study aimed to identify the spawning sites in the Rhine system. We conducted a tank experiment and kept shad larvae in water from four sub-catchments of the Rhine system. We analysed trace substance concentrations in water samples and the microchemical composition of otoliths from reared larvae. Using a random forest model, we were able to correctly attribute the larvae to the sub-catchment where they were raised based on elemental/ratio (Sr/Ca) and strontium isotopes (87Sr/86Sr). From the 66 allis shad caught in the Rhine system between 2017 and 2020, seven individuals (11%) were identified as being stocked. Of the 59 remaining individuals that came from natural reproduction, 37 were attributed to the Rhine, 7 to the Neckar and 4 to the Lippe sub-catchments with high certainty. We also observed allis shads dispersed in adjacent catchment areas and a homing behaviour. A total of 27% of the adults (n = 9) and 8% of the juveniles (n = 2) were assigned to any of the four sub-catchments included in our model, suggesting the need to expand the model and include additional sub-catchments to cover all spawning sites in the Rhine system and adjacent catchments.