Introduction: Antibiotic resistance is the leading cause of Helicobacter pylori infection therapy failure. Statistics on antibiotic resistance and genetic mechanisms of resistance are lacking in Oman, which is important for choosing the right medications to eradicate H. pylori. This study aims to investigate the antimicrobial resistance of clarithromycin, amoxicillin, levofloxacin, metronidazole, and tetracycline phenotypically in H. pylori. Moreover, this study focuses on the molecular characterization of multi-resistant isolates by Illumina whole genome sequencing. Methods: Gastric biopsies were collected through endoscopy from patients with gastroduodenal symptoms and screened for H. pylori using the rapid urease test; CLO test. Three hospitals were included in this study: Sultan Qaboos University Hospital (SQUH), Royal Hospital (RH) and Creativity Medical Centre (CMC). The positive biopsies were transferred to the microbiology laboratory in a transport media. Phenotypic testing of antibiotic susceptibility was performed using two methods; broth microdilution and E-test strips for six antibiotics (clarithromycin (CLA), amoxicillin (AMX), metronidazole (MTZ), tetracycline (TET), rifampicin (RIF) and levofloxacin (LEV)), The DNA was extracted for whole-genome sequence (WGS). Data obtained from WGS was analyzed for the genotype of antimicrobial resistance, virulence factors, Multi-Locus Sequence Typing (MLST), and phylogenetic relatedness of the strains. Results: The total number of biopsy samples collected from three different centers according to inclusion criteria was 169 but the total number which was successfully growing in the growth culture was only 23. Antibiotic susceptibility testing by using two methods, broth microdilution and E-test, showed that the highest levels of resistance was in MTZ (93.3%) and 100% respectively. There was agreement between the two methods in all antibiotics except metronidazole. For a 95% confidence interval, the limits of agreement for six antibiotics were as follows: (-42.6,18.4) for CLA; (-9.2,28.4) for AMX; (-94, -8.4) for TET; (-8.4,12.6) for MTZ; (-6.5,20.7) for RIF; (-.8,1.6) for LEV. None of the isolates were resistant to rifampicin according to previous study MIC instead of EUCAST guidelines. WGS data was used to characterize the genotype of resistant isolates by searching for point mutations in resistance-conferring genes, Among the 20 strains, 33.3% strains showed resistance to CLA by E test and with mutations in 23S rRNA (A2146G, A2147G, C1707T and A2144G). Amoxicillin phenotypic resistance in 66.6% of isolates was associated with point mutations in pbp1, pbp2, and/or pbp3. None of the strains were sensitive to MTZ by both methods with mutations in rdxA (T31E, R90K, A118T, A67V, C49T, R16C, D59N, H97T, H97Y, K64N, P106S and S108A) and frxA (Y62D, A16T, A15V, A85V, M126F, A70G ndV7I). All isolates were genotypically susceptible to tetracycline, although one isolate was resistant phenotypically. Among the 65.7% RIF-resistant strains, mutations in ropB were in the positions K2068R, I837V, and Q2079K whereas only 33.3% showed phenotypic resistance. Strains with mutations in gyrA (N87K, D91G andV172I) were resistant to LEV (20%). The genotype and phenotype agreement in the six antibiotics tested was statistically insignificant (p=1). MLST analysis showed that twelve strains belonged to ST 3120, one isolate belonged to 3104 and seven isolates were novel. The phylogenetic tree shows that these strains were very closely related, which suggests that the strains circulating in the Omani population are very similar. The most significant H. pylori virulence genes are presented in this study along with key findings. The presence of virulence genes can affect the patients and increase the risk of gastritis, peptic ulcers, inflammation, and gastric cancer as shown in this study. Conclusion: In summary, our findings indicate that H. pylori demonstrated a high significant resistance to metronidazole, followed by clarithromycin, and amoxicillin with contributing point mutations. Furthermore, antibiotic susceptibility and molecular testing by WGS are essential to guide appropriate and population-specific antibiotic therapy.