With 3,480 E. coli strains isolated from the Yeongsan River basin, South Korea, correlations between phylogenetic groups and horizontal fluorophore enhanced rep-PCR (HFERP) genotypes were examined, and environmental factors affecting E. coli phylogenetic groups in the river water were determined. Interestingly, multidimentional scaling (MDS) analyses based on HFERP DNA fingerprint data indicated that E. coli in phylogenetic groups A and B1 were uniquely clustered. Results of self-organized maps (SOMs) analyses also indicated that E. coli phylogenetic groups were seasonally affected by water temperature, with greater occurrences of phylogenetic groups A and B1 in low and high temperature seasons, respectively. The presence of E. coli in phylogenetic groups A and B1 were inversely related. Furthermore, redundancy analysis (RDA) revealed that phylogenetic group B1 correlated positively with temperature, strain diversity, and biochemical oxygen demand (BOD) but negatively with phylogenetic group A. Results of this study indicated that while E. coli strains could be clustered based on their genotypes and environment conditions, their phylogenetic groups did not change in relation to the same conditions. The distributional differences of phylogenetic groups among E. coli populations in different environments may be caused by different genomic adaptability and plasticity of E. coli strains belonging to each phylogenetic group. Although several previous studies have reported different E. coli ecological structures depending on their origins, this study is a first description of the specific environmental factors affecting E. coli phylogenetic groups in river water.