Temporal variability in the gastrointestinal flora of animals impacting water resources with fecal material can be one of the factors producing low source identification rates when applying microbial source tracking (MST) methods. Understanding how bacterial species and genotypes vary over time is highly relevant when the fecal material used to create a source library is collected under very different seasonal conditions than the environmental sample. Our objective was to identify and compare the temporal and spatial variability of fecal indicator bacteria from a specific host in manure and water samples and evaluate the implications of such variability on microbial source tracking approaches and applications. We selected Enterococcus as the model fecal indicator, given the supposedly high specificity of some of the species of this genus to the host organism. Cattle was chosen as the model host organism because of the documented high impact that cattle has on impairment of surface waters. The sites studied were located at a farm where cattle have unrestricted access to the stream. Enterococci were isolated monthly from water and manure samples using membrane-Enterococcus Indoxyl-Beta-D-Glucoside agar (mEI) as described in EPA method 1600. The isolates were identified using a multiplex PCR procedure that targets the genus and the species-specific gene superoxide dismutase. Eight species were identified in cattle manure, of which E. casseliflavus (37%), faecium (22%) and hirae (18%) were the most abundant. Nine species were identified in stream samples with E. faecalis (43%), casseliflavus/flavescens (34%), and hirae (11%) being the most abundant. September exhibited the highest species abundance in manure samples while March had the highest species abundance in stream water samples. E. assini and E. malodoratus were only detected in manure samples, but were not detected in water samples. In contrast, E. durans, gallinarum and sulfureous were only isolated from the stream samples. In general, the enterococci distribution pattern and species richness found in manure samples did not correlate with those found in the stream samples at the individual species level. However, cluster analysis revealed strong seasonal and spatial variability of groups of enterococci, and indicated that some clusters that seem specific to manure can be found in the water only during certain seasons. In addition to the enterococci library development, 16S rDNA host-specific Bacteroides markers were also applied to the water samples. The results indicate that data obtained with the Bacteroides markers (BM) generally agreed with the enterococci data showing higher occurrence of the cattle BM in areas under obvious cattle impact. However, no seasonality was identified in conjunction with any of the BMs used. In addition, the cow marker was also detected at an upstream-of-the-farm location that was not under obvious cattle influence. This study suggests that in order to increase the validity of MST methods, it is necessary to consider temporal variability when designing the sampling scheme of the source material and constructing source libraries, and increase the specificity and field testing of DNA-based markers.