Background: The evident multidrug resistance (MDR) associated with Helicobacter pylori (H. pylori) is a serious public health problem. Multidrug resistance was defined as resistance to at least one antimicrobial agent in three or even more antimicrobial classes. The hefA gene encodes an active efflux process against antimicrobials, and its expression in H. pylori may contribute to the upsurge of resistant strains.  Aim of the study: Detection of MDR H. pylori among endoscopic isolates and examining the role of hefA gene expression in the occurrence of MDR H. pylori. Methods: Our research involved 40 H. pylori endoscopic isolates using conventional microbiological methods. We identified MDR in H. pylori isolates using E testing procedures for metronidazole, ciprofloxacin, amoxicillin as well as clarithromycin. We detected the hefA gene expression among the isolated H. pylori by real-time PCR.  Results: out of 40 isolates of H. pylori, 13 (32.5%) were MDR and 27 (67.5%) were not MDR. We found hefA gene in MDR H. pylori isolates with total (mean ± SD) equal to 7.055385±2.591111, but in non-MDR H. pylori isolates were with total (mean ± SD) equal to 2.591111±0.720189. There was a statistically significant difference in hefA expression levels among both MDR H. pylori isolated strains and non-MDR ones; the p value was 0.001. Conclusion: The hefA gene expression in H. pylori plays an important role in the emergence of MDR H. pylori strains as one of the genes encoding for efflux pump mechanisms.