Introduction: Uropathogenic Escherichia coli (UPEC) remains the most common cause of urinary tract infections (UTIs). They account for over 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. E. coli strains have been found to belong to evolutionary origins known as phylogenetic groups. In 2013, Clermont classified E. coli strains into eight phylogenetic groups using the quadruplex PCR method. The aim of this study was to identify the phylogenetic groups of UPEC strains in Uganda using Clermont’s quadruplex PCR method and to assess their antibiotic susceptibility patterns in Uganda. Methods: In this cross-sectional study, 140 stored uropathogenic E. coli isolates from the Clinical Microbiology Laboratory, Department of Medical Microbiology, College of Health Sciences Makerere University were subjected to phylogenetic typing by a quadruplex PCR method. Antimicrobial susceptibility testing was performed by disk diffusion method according to Clinical & Laboratory Standards Institute (CLSI) guidelines. Phenotypic detection of extended-spectrum beta-lactamase, AmpC and carbapenemases was done according to CLSI guidelines and Laboratory SOPs. Results: Phylogenetic group B2 (40%) was the most predominant, followed by A (6.23%), clade I and II (5%), D and E (each 2.14%), B1 (1.43%) and F and C (each 0.71%). The most common resistant antibiotic was trimethoprim-sulphamethoxazole (90.71%) and the least was imipenem (1.43%). In total, 73.57% of isolates were multi-drug resistant (MDR). Antibiotic resistance was mainly detected in phylogenetic group B2 (54%). Conclusions: Our findings showed the high prevalence of MDR E. coli isolates, with the dominance of phylogenetic group B2. About 9% of E. coli isolates belonged to the newly described phylogroups C, E, F, and clade I and II.