Better understanding of Escherichia coli population dynamics and genetic variability in the secondary habitat is essential to improve fecal contamination monitoring and contamination pathway characterization. In this study, water samples were collected monthly over a one-year period at eight locations in the Catoma Creek watershed, a mixed land-use watershed in Central Alabama. E. coli concentrations varied from 17 to 12,650 CFU/100 ml and were well correlated with stream flow rates. Repetitive sequence-based PCR DNA fingerprinting was used to generate 271 unique DNA fingerprint patterns from 502 E. coli isolated from water samples. Cluster analysis showed an overall similarity of 32.8% across all DNA fingerprints. Multivariate analysis of variance (MANOVA) showed that E. coli genotypes had a tendency to cluster according to season and stream flow rather than sampling sites. MANOVA of a subset of data within a given season and flow rate, however, revealed some geographical differentiation between urban and rural sampling sites. The results indicate that genetic diversity of E. coli populations was not only high in the secondary habitat but also varied with season, flow conditions and, to a lesser extent, sampling location. To our knowledge, this is the first report relating E. coli genotype to stream flow.