Urinary Tract Infections (UTIs) during pregnancy are among the most common infections worldwide and can lead to poor perinatal and maternal outcomes, especially in developing countries and requires medical treatment as soon as it is detected. Virulent and resistant strains of Escherichia coli are the most important causative agents for UTIs. The present investigation aimed to detect virulence factors of Uropathogenic Escherichia coli (UPEC), including pap, fim, sfa, aer and hly genes in isolates collected from symptomatic and asymptomatic pregnant females by multiplex polymerase chain reaction (PCR) assay and determine their correlation with antibiotic resistance patterns. Two hundred urine samples collected from pregnant females with or without symptoms of UTI were admitted to antenatal clinic at Obstetrics and Gynecology Department of Al-Zahraa University Hospital in Cairo. Out of 200 cultured urine samples, the cultures that showed ≥ 10 5 CFU/ml were identified to have significant growth. Found significant bacteriuria was detected in 61 samples and these samples selected for further analysis in this study, the overall prevalence of UTI among pregnant females was (30.5%). Symptomatic pregnant females with UTI were (48.5%) more than asymptomatic (11.3%). The most frequently isolated species was S. saprophyticus (35.0%), followed by E. coli (26.2%), S .aureus (19.4%), C .albicans (5.8%), S. epidermidis (2.9%) and each of K. pneumonia, K .oxytoca, P .vulgaris, E.fecalis, Bacillus spp were (1.9%), while P. auroginosa had the least percentage of isolation (1.0%). Antimicrobial susceptibility pattern was done for 27 UPEC isolates showed the highest level of resistance (100%) against β-lactams as (Ampicillin, Penicillin and Ceftriaxone), nitrofurantoin, and ampicillin/sulbactam, while the lowest level of resistance (66.7%) against Cotrimoxazole. Multiple drug resistance (resistance to two or more drugs) was observed in (100%) of the UPEC isolates. The higher level of resistance to antibiotics was observed in symptomatic bacteriuria more than asymptomatic. Among two predominant uropathogens, E. coli showed complete resistance up to 5 different antibiotics of total 11 antibiotics (45.5%) while S. saprophyticus showed complete resistance (18.2%), so E. coli showed higher virulence and resistance than S. saprophyticus to cause UTI, so subjected to Multiplex PCR to detect its virulence factors, which included pap, fim, sfa, aer and hly genes The adhesive fimH gene was the most frequent in UPEC isolates (92.6%). These virulence genes detected from total UPEC isolates of symptomatic cases were higher than asymptomatic. On the other hand, UPEC strains carrying the virulence genes were more resistant to the antibiotics used, so these observations confirm the important role of virulence genes of UPEC in in existence of symptoms of UTI and the drug resistance.