Urinary tract infections (UTIs) are frequently encountered bacterial infections treated with antibiotics. Extraintestinal Escherichia coli is responsible for majority of the UTIs. Infections associated with particular clonal lineages such as fluoroquinolone resistant ST-131 are becoming increasingly challenging to treat. The notion that the acquisition of MDR factors have the fitness cost associated in terms of compromise on virulence potential has led to the scrutiny of the MDR strains of UPEC across different continents. However, there is very scarce knowledge about the clonal types and genetic characteristics of UPEC strains associated with UTIs in Pakistan region. In this study, we scrutinized genetically distinct MDR UPEC strains for number of virulence factors, however particular focus was the role of iron acquisition system in invasion process. Furthermore, by Whole Genome Sequencing genetic blueprint of 20 different UPEC strains was assembled. PCR screening of 155 UPEC isolates, confirmed presence of fimH (100%), iutA (55%), feoB (49%), papC (48%), papGII (45%), kpsMTII (26%), papEF (24%), fyuA (24%), usp (14%), papA (13%), sfa/foc (13%), hlyA (12%), afa (10%), cdtB (7%), papGI (4%), papGIII (4%), kpsMTIII (3%) and bmaE2 (1%). Certain VF factors such as cell surface hydrophobicity, mannose resistant hemagglutination, papGII, sfa/foc and feoB were frequently distributed among MDR strains, while these strains were resistant to the major frontline antibiotics including trimethoprim and cephalosporins. WGS was performed using next generation MiSeq and HiSeq 2500 platforms. SPAdes v.3.11 was used for the De novo assembly of the reads, whereas genomic features were determined using PATRIC and RASTtk. Mutants were produced by λ-Red recombination and invasion assays were performed to determine the virulence potential in-vitro. Among different sequence types, VF were uniformly distributed, whereas, some VF such as papEF, sfa/foc, fyuA and iutA occurred frequently among ST131 strains. Selected MDR-virulent strains were tested for their invasion ability which was dependent on the presence or the absence of different iron acquisition genes such as enterobactin (entABCDEFH, fepA), yersiniabactin (ybtPQirp1irp2, fyuA), aerobactin (iucABCD, iutA), and the heme uptake systems (hmuRSTUV). Strains having all three siderophores along with the heme uptake systems were significantly more invasive when compared to their counterparts having either of three siderophore genes. The gene knock out experiments confirmed that the loss of yersiniabactin and aerobactin has the most profound effect on the invasive ability of UPEC. WGS sequencing of the MDR virulent clone of ST131-O25b-H30 lead to the identification of an array of molecular factors, including blaCTX-M-15, blaOXA-1, blaCMY-2, sul2, catB, dfrA17, mph (A) a class 1 integron, 77 insertional sequences (IS elements), a Tn3-like transposon, multiple virulence markers and 7 intact prophage loci. Likewise, WGS of another clone, ST38-O1:H15 identified blaTEM-1, CMY-2, sul1, sul2, dfrA17, tetA, mphA and mobile elements int1, two transposons, 30 insertion sequence elements, one integrative conjugative element, four plasmids and five prophages. A comparative genomic analysis of twenty MDR-virulent UPEC isolates identified multitude of VF, resistance genes, plasmids and prophages. Certain VF (papI, papC, papE, papH, RTX, cnf) resistance genes (blaCTXM-15, blaOXA-1, catB, dfrA17) and plasmid incompatibility groups (IncFIA and IncI1) were significantly associated with the clonal group ST131. In conclusion, this study confirms an array of VF and MDR factors in UPEC strains. Moreover, invasion assays confirmed that the invasion ability of tested strains directly depend on their ability to sequester iron and the loss of siderophore genes significantly impairs invasion ability. WGS and comparative genomic analysis of MDR UPEC strains revealed their diverse virulence and MDR profile which indicates a successful acquisition of MDR factors by the virulent UPEC strains circulating in this part of the world. Among these the predominant clonal group ST131 showed diverse virulence and MDR profile postulated as a significant fitness advantage to these strains.