Previous studies have shown that Escherichia coli can be isolated from non-polluted rivers and from bromeliad axilae in pristine areas of tropical rain forests. Finding E. coli in pristine environments is unusual because this bacterium is thought to only survive in the gut of warm-blooded animals and thus its presence should indicate recent fecal contamination. The aims of this study were 1) to determine if E. coli is part of the native soil microbiota in tropical rain forests and 2) to determine if genetic heterogeneity exists among E. coli populations. High concentrations of total coliforms (104–105 cells per 10 g of soil dry weight) and low concentrations of thermotolerant coliforms (101–102 cells per 10 g dry soil, the majority of these were found to be E. coli) were detected. PCR using uidA-specific primers was done on DNA purified from E. coli isolates and the resulting amplicons analysed by denaturing-gradient gel electrophoresis (DGGE). Out of several hundred isolates, mixtures of nine different amplicons were consistently observed. The different patterns of DGGE observed indicate that the E. coli populations in these pristine soils are genetically heterogeneous. Fecal and environmental E. coli isolates were also analysed by pulsed-field gel electrophoresis (PFGE) which showed high DNA sequence variation among the E. coli isolates. Because of these differences in the genomes, PFGE did not allow grouping of environmental versus human isolates of E. coli when compared side to side. The apparent genetic polymorphisms, as a result of genetic heterogeneity, observed in isolates from the same pristine site indicate that source tracking may be difficult to carry out using E. coli as the target organism.