Background: Urinary Tract Infections (UTIs) caused by extended spectrum beta lactamase (ESBL) producing E.coli in pregnancy is a serious health problem increasing pregnancy–related complications and continue to be a challenge for choosing the suitable therapy since they may exhibit coresistance to many other classes of antibiotics. Objectives: to ascertain the genotypic and phenotypic characteristics of (ESBLs) producing E. coli from pregnant females attending the Obstetrics and Gynecology Department in Benha University hospitals. Methodology: 100 urine samples of pregnant females were presumptively identified using standard bacteriological methods. Positive cultures were tested for antibiotic sensitivity. Production of ESBL was confirmed by double disc synergy and MALDI-TOF MS assay. Loop mediated isothermal amplification (LAMP) was further applied for the ESBL gene genotypic detection. Results: 34(82.93%) women out of 100 were colonized by ESBL producing E.coli. The rates of antibiotic susceptibility of E.coli isolates were 53.66% to Ampicillin, 43.9% to pipracillin, 30.9% to gentamicin, 43.9% to aztreonam, 21.9%, 39% and 17% to ceftriaxone, cefotaxime and cefpodoxim respectively. Concerning genotypic detection of ESBLs genes; CTX M-1 was (29.2%), followed by CTX M-2 (4.9%). Conclusion occurrence of ESBL synthesis among E. coli isolates is high. Periodical detection and checking of antimicrobial susceptibility of ESBL isolates in pregnant women, and the use of effective antimicrobial drugs are recommended to decrease the risk of high antibiotic resistance rate and reduce potential subsequent complications.