High concentration of fecal indicator bacteria is the most common cause of surface-water impairment in Alabama. Catoma Creek in Montgomery County has been included on the state Section 303(d) list due to elevated concentrations of fecal coliform bacteria and organic enrichment. Forest, Agriculture, and urban land uses represent 54.5%, 36.2%, and 9.3% respectively, of the watershed. This study was conducted to monitor the fecal contamination of the Catoma Creek watershed and to determine the genetic variability of E. coli isolates obtained from stream water. Water samples were collected monthly at eight locations in the watershed for a period of one year. E. coli was enumerated using the modified m-TEC media. Data showed that E. coli concentrations varied from 18 to 12,650 CFU/100 ml, with 70% of the samples exceeding the EPA criterion for swimming water. There was a high correlation between flow rates and E. coli concentrations. The rep-PCR DNA fingerprint method was used to develop DNA fingerprint patterns for 503 E. coli isolated from water samples. PCR reactions using BOXAIR primer generated between 5 and 28 product bands and 271 unique fingerprint patterns were found. Cluster analysis showed an overall similarity of 32.8% for all the DNA fingerprints. Discriminant analysis showed that E. coli fingerprints had a tendency to cluster according to seasons and stream flow rather than sampling sites; however, the clusters were not tight. The results indicate high genetic diversity of E. coli isolated from stream samples. Large numbers of E. coli isolates are needed to represent potential host sources when a library-based approach is used for bacterial sources tracking.