Biodegradation is one of the most important processes influencing the fate of organic contaminants in the environment. Quantitative understanding of the spatial variability in environmental biodegradation is still largely uncharted territory. Here, we conducted modified OECD 309 tests to determine first-order biodegradation rate constants for 97 compounds in 18 freshwater river segments in five European countries: Sweden, Germany, Switzerland, Spain, and Greece. All but two of the compounds showed significant spatial variability in rate constants across European rivers (ANOVA, P < 0.05). The median standard deviation of the biodegradation rate constant between rivers was a factor of 3. The spatial variability was similar between pristine and contaminated river segments. The longitude, total organic carbon, and clay content of sediment were the three most significant explanatory variables for the spatial variability (redundancy analysis, P < 0.05). Similarities in the spatial pattern of biodegradation rates were observed for some groups of compounds sharing a given functional group. The pronounced spatial variability presents challenges for the use of biodegradation simulation tests to assess chemical persistence. To reflect the variability in the biodegradation rate, the modified OECD 309 test would have to be repeated with water and sediment from multiple sites.