The development of methodology to differentiate mixed populations of Escherichia coli in the secondary habitat might improve monitoring of fecal pollution indicators and facilitate the development of strategies to mitigate bacterial pollution. The objective of this study was to determine the ability of denaturing gradient gel electrophoresis (DGGE) to differentiate mixed assemblages of E. coli in the natural environment. After confirming the identity of 184 environmental bacterial isolates as E. coli, each was subjected to polymerase chain reaction (PCR) of the β‐glucuronidase gene (uidA) followed by DGGE fingerprinting. The ability of DGGE to discriminate individual isolates at the strain level was determined by comparing fingerprints to those resulting from a standard, library‐dependent fingerprinting method, BOX‐PCR. Computerized analysis of fingerprints indicated that DGGE and BOX‐PCR identified 15 and 21 unique phylotypes respectively. Rank–abundance plots comparing the numerical distribution of unique E. coli phylotypes detected by both methods revealed no difference in resolution at the population level. In water and sediment samples from two beaches, DGGE effectively distinguished indigenous E. coli populations with an average rate of correct classification (site‐based) of 83%. Denaturing gradient gel electrophoresis of uidA genes isolated and PCR‐amplified from environmental samples appears to be an effective tool to differentiate unique E. coli populations and should be useful to characterize E. coli dynamics in the secondary environment.