Background:  Escherichia coli (E. Coli) can cause septicemia, which is a deadly infection. Furthermore, the options for eradication have been restricted by the presence of virulence factors and multidrug resistance.  This study aimed to detect phylogenetic groups, assess clinically important virulence genes, and determine the genetic relatedness of isolates. Methods: A total of 100 isolates of multidrug-resistant E. Coli were taken from septicemia. The disk diffusion method was used to test for antibiotic susceptibility. Polymerase chain reaction (PCR) was used to screen for virulence genes, carbapenemases, and extended-spectrum β-lactamases. Additionally, serogrouping and phylogenetic grouping were examined. Pulsed field gel electrophoresis was used for the genetic classification (PFGE). Results: Tetracycline, ceftazidime, and cefepime resistance was present in all isolates. There were 56, 55, and 43% of patients resistant to imipenem, meropenem, and gentamicin, respectively. The blaCTX-M1 (51%), blaSHV (64%), blaIMP (33%) and blaOXA-48-like (12%) genes were present in the isolates. In 22%, 17%, 36% and 25% of cases, the phylogroups A, B1, B2, and D were found respectively. O1 accounted for 53% of the total serogroups, with O25 at 14% and O75 at 11%. The virulence genes csgA, iutA, HlyA, traT, and KpsMII were present in all of the isolates. Of the strains carrying blaCTX-M1, 80% (p<0.0001) had the O1 serogroup and 62% (p<0.001) belonged to the B2 phylogroup. Five clusters (A–E) with an 80% cut-off and no genetic relationship were shown by the PFGE. Conclusion: It is imperative to implement surveillance and appropriate control measures to stop the spread of drug-resistant and virulent E. Coli from septicemia.