Current management of gastric inflammation involves the eradication of Helicobacter pylori. However, the effectiveness of commonly used antibiotics against H. pylori infection has decreased due to antibiotic resistance. Phenotypic-based diagnostics are laborious and finding the cause of resistance can be difficult. Therefore, early detection and understanding of the underlying mechanism of this resistance are necessary. This study evaluated the mutations in the genes related to the Antimicrobial Resistance (AMR) of the clinical isolates from Bangladeshi subjects. Whole-genome sequencing was performed on 56 isolates and the genes (such as pbp1a, rdxA, ribF, fur, gyrA, gyrB, 23S rRNA, and infB) were extracted. The reads were assembled, and the SNPs were extracted by the latest pipeline for antibiotic mutation analysis, ARIBA. The mutations and the association with the antibiotic phenotypes were evaluated using Fisher’s exact test. In this study, the clarithromycin resistance rate was high, 39.3% (22/56), with the median MIC 24 mg/L ranging from 2 to 128 mg/L. The mutation of A2147G was significantly associated with resistance (p = 0.000018) but not in locus A2146G (p = 0.056). Levofloxacin also posed a high resistance. We observed that the mutation of D91N (but not D91Y) (p = 0.002) and N87K (p = 0.002) of gyrA was associated with levofloxacin resistance. Mutations in locus A343V (p = 0.041) of gyrB also showed a significant association. Meanwhile, in the pbp1a gene, several mutations might explain the resistance; they were G594fs (p = 0.036), K306R (p = 0.036), N562Y (p = 0.0006), and V45I (p = 0.018). The prevalence of metronidazole was exceptionally high (96.4%), and numerous mutations occurred in rdxA genes, including the truncation of genes. These results imply that the mutation in genes encoding the target protein of antibiotics remains the critical resistance mechanism in H. pylori.