The multidrug-resistant (MDR) Escherichia coli, particularly β-lactamase producing strains, has become a global health challenge, where wastewater systems, specially from hospitals, serve as critical reservoirs for the dissemination of resistance genes. The objectives of the study were to investigate the whole genome sequence diversity and genetic variations focusing on their evolutionary relationships, genetic similarity, and single nucleotide polymorphisms (SNPs) of pathogenic β-lactamase producing E. coli strains. A total of four β-lactamase producing E. coli strains, from differently located tertiary care hospitals, were included in this study. A heatmap of genetic similarity revealed near-identical genetic makeup among the strains. A number of genes including AcrAB-TolC, β-lactamases, and resistance determinants such as glpT, gyrA, msbA, and tet(M) were identified in these strains. However, the presence of virulence genes of the aerobactin synthesis gene (iucA, iutA) and type III secretion systems (espX1, espX4, espX5) in the strain has the potential for pathogenicity. These resistance genes were identified at the genomic level; however, their transcriptional expression was not evaluated and the detection of virulence-associated genes suggests that the isolates have the potential to cause disease and pathogenicity. These findings provide insights into the whole genome sequence diversity of E. coli in urban based tertiary care hospital wastewater, emphasizing the genetic variation and plasmid diversity in these E. coli strains, which may have implications in public health and microbial ecology of the environment.