Organic molecules that contain conjugated aromatic constituents have the potential to fluoresce. Both natural and anthropogenic organic matters may contain fluorescent molecules, and river and groundwater organic matters can be understood as a complex mixture of fluorescent and nonfluorescent organic molecules. The investigation of pollution in rivers and groundwaters, therefore, requires the differentiation of multiple fluorescent molecules from multiple sources. The fluorescence spectra of both natural and pollutant organic matters are increasingly well known. Fluorescent pollutants in rivers and groundwaters are typically identified by high levels of fluorescence in the shortwave ultraviolet spectra associated with high levels of microbiological activity and biochemical oxygen demand (BOD); the presence of polycyclic aromatic hydrocarbons from landfill leachates or petroleum products; or the presence of fluorescent whitening agents (FWAs) from industrial, landfill, or sewerage pollution. These fluorescence signals can be distinguished from natural organic matter fluorescence by analyzing either of the differences in spectral properties, often using multiway analysis such as parallel factor analysis, or the investigation of their sensitivity to microbial or photodegradation. Examples of the investigation of pollution in rivers and groundwaters by fluorescence using both laboratory instrumentation and in situ probes are discussed.