Abstract Aim To investigate the phenotypic and genomic features of three multidrug-resistant (MDR) clinical mucoid and non-mucoid uropathogenic Escherichia coli (UPEC) strains to understand their antimicrobial resistance, biofilm formation, and virulence in urinary tract infections (UTIs). Methods and Results The UPEC strains A5, A10, and A15 were isolated from two UTI patients. Phenotypic assays included colony morphology, antibiotic susceptibility, motility, and biofilm formation. Whole genome sequencing (WGS) identified resistance genes, virulence factors, sequence type (ST), serotypes, plasmid types, and gene variations. While strains A5 and A10 were mucoid, A15 was non-mucoid. Although obtained from a single patient, A10 formed biofilm while A5 did not. Both strains harboured blaNDM-5 and blaCMY-145, while A15 carried blaCTX-M-15. Genomic variations in A10 included mutations in fdeC and chiA (biofilm genes), while A15 showed changes in fyuA, pdeR, pdeF, and waaZ. A comparative genome analysis of our strains with Indian UPEC isolates revealed that presence of blaCMY-145 in strain A5 and A10 may be rare. Conclusions Despite the genetic similarity, UPEC strains exhibit distinct phenotypes possibly due to variations in inducible conditions including host and environmental factors. These differences highlight the multifactorial nature of UPEC pathogenicity and the influence of both genetic and host-related factors.