Objective: This study aimed to determine the prevalence of Metallo-β-lactamases (MBLs) among clinical E. coli isolated from North of Palestine. Methods: A total of 79 of E. coli isolates were recovered from hospitals in North of Palestine during February-July 2015. A multiplex PCR was used to determine the preva-lence of MBLs among these isolates. Results: Results showed that the prevalence of MBLs was 87.4%. The spm gene was the most prevalent (86.1%) among these isolates. According to the geo-graphical distribution, results showed that the prevalence of MBLs among E. coli isolates recovered from Thabet Hospital-Tulkarm was 75.9%. The Spm gene was the most common (72.4%) among these isolates, while the Imp gene was detected in 41.4% of isolates. For isolates recovered from Jenin Hospitals, the prevalence was 94% and spm gene was detected in all MBL producers, 2 other genes were detected, Imp and Sim, and the prevalence was 12% and 2%, respectively. A total of 26.1% of MPL E. coli producers carried 2 genes. Conclusions: Our results showed high occurrence of MBLs among E. coli isolates in Palestine. Based on these results we recommend the continuous monitoring and surveillance of the prevalence, proper control and pre-vention practices and effective antibiotic use will limit the further spread of MBLs producing isolates within hospitals in this country. Prevalence of MBL producing microorganisms has not been investigated previously.