Given the interest in Bacteroides species as microbial source tracking (MST) markers, and the limited knowledge of the survival of Bacteroides species in the environment, here we examine the survival of Bacteroides fragilis, B. thetaiotaomicron, and environmental species of Bacteroides by use of culture techniques and molecular tools. Two kinds of experiments were performed: (i) on-site experiments, in which bacteria were exposed to changes in the levels of several environmental parameters in a river, and (ii) microcosm assays in the laboratory, with controlled temperatures. On-site experiments showed different survival patterns for the cultivable Bacteroides strains. B. fragilis die-off rate was strongly affected by the combined effect of high temperatures and grazing predators, which were more active under warmer conditions. However, the survival rates of cultivable B. thetaiotaomicron and environmental Bacteroides spp. were more affected by dissolved oxygen (DO) concentration in water. Environmental Bacteroides strains survived longer than either type strain, due to better adaptation to environmental conditions. However, the period of their survival was shorter than that observed for fecal coliforms and enterococci, suggesting Bacteroides species as markers of recent fecal pollution. The total Bacteroides species were detected by molecular techniques throughout the experiment in winter, but they were detected on only two or three days in the summer. This indicates that temperature is the main factor affecting DNA degradation, regardless of species. The use of microcosms in the laboratory also pointed to temperature as the main factor affecting Bacteroides survival, regardless of species. However, the conditions in the laboratory may mask the effects of the environmental factors and their interactions. The observed variability in die-off rate as a function of the species analyzed, the experimental conditions, and the methodology used should be taken into consideration in future persistence studies.