Extra-Intestinal Escherichia coli (ExPEC) are important cause of Urinary Tract Infections (UTIs) and systemic infections. The purpose of this study was to investigate numerous ExPEC bacterial isolates for phenotypic virulence characteristics including hemolytic activity and resistance pattern and to observe their association with genetic traits via Polymerase Chain Reaction (PCR). A total of 367 ExPEC isolates were collected from patients admitted in Khyber Teaching Hospital (KTH) Peshawar, Pakistan. Standard techniques were used for identification of isolates, determination of hemolytic potential and antimicrobial susceptibility testing. PCR was used for screening of virulence genes using specific primers. A total of 367 ExPEC isolates were characterized, among which 62.7, 24.3, 7.1 and 6% were isolated from urine, pus, sputum and wound specimens, respectively. Majority of the isolates (82.8%) were hemolysin positive. Multi drug resistance pattern was shown by 41% of the isolates and harbored at least one virulence gene (71.7%), of which sat was the most prevalent (64.3%). The highest resistance was found to cefotaxime (99.2%), ampicillin (97.5%) and aztreonem (89.6%). 15 different virulence genes combinations were observed in the current study. A total of 16 virotypes (15 of positive virulence genes and one of no virulence gene) were observed in the current study. The current investigation showed a high prevalence of sat and hlyA genes among ExPEC isolate, suggesting a role of these genes in the pathogenesis of ExPEC.