Methylxanthines are commonly recommended as anthropogenic markers, yet possible diffuse sources are largely unaccounted for. In this study, eight methylxanthines and xanthine were determined jointly in an entire river system for the first time. Overall, 49 samples were taken from the River Wietze (Lower Saxony, Germany) and its tributaries in four sampling campaigns. In addition, consecutive rainwater samples as well as influent and effluent samples from the largest adjacent municipal wastewater treatment plant (WWTP) were taken. All samples were enriched using SPE and analysed via LC-MS/MS. For xanthine, the standard addition method was applied. All investigated compounds were found in concentrations ranging from some dozen to some hundred ng L⁻¹. For isocaffeine, 7-methylxanthine, and xanthine, measured concentrations in the River Wietze are the first available concentrations in surface water. Mass loads displayed a high seasonal variability and up to 144 g day⁻¹ of methylxanthines were discharged into the receiving river. Municipal WWTPs were found to be an important and constant point source of all compounds. However, the high dependence of mass loads on discharges in the river as well as the detection of most methylxanthines at the blank site and in rainwater samples suggest additional diffuse introduction pathways. Solely the occurrence of isocaffeine in the River Wietze was found to be exclusively attributable to municipal wastewater discharge. Therefore, while caffeine and its degradation products are unsuitable for tracking the presence of treated or untreated wastewater in fluvial systems, isocaffeine can be recommended as a marker for the discharge of treated wastewater.