Escherichia coli is widely used to indicate faecal contamination in water, as it is easily identified, a ubiquitous inhabitant of the intestinal tract of humans/other mammals with limited persistence outside a host (<3 days). Strains of E. coli can be partitioned into subgroups (A-B1-B2-D-C-E-F and clade I-V) (1). The clonal composition of E. coli inhabiting most host populations is not reflected in E. coli isolated from aquatic environments, in the latter phylogroup B1 predominate (2). Previous studies suggest certain B1 strains have enhanced persistence in water (>12 days) relative to other E. coli strains (2). Although B1 strains are uncommonly detected in human faeces, they are common in other host groups (carnivorous mammals and birds) (3). A recent study demonstrated a lineage of B1 strains (clonal complex 87) represents a predominately host-associated lineage (4). The relative abundance of this lineage in water is unknown, nor is it known if particular B1 strains predominant in aquatic environments. Consequently, whole genome sequencing was performed on 118 B1 strains (repeatedly isolated from the aquatic environment across eastern Australia) together with 74 diverse faecal B1 strains. Phylogenetic analysis revealed two distinct lineages of B1 strains that predominately consisted of environment strains. These lineages lack antibiotic resistance determinants and harbour minimal virulence genes associated with intestinal and extra-intestinal disease. Subsequently, they appear to be members of autochthonous populations of E.coli that inhabit diverse aquatic environments. This study enhances our understanding of persistent B1 E. coli strains in water and demonstrates their existence confounds E. coli’s use a water quality indicator.