Background: Helicobacter pylori is an important human pathogen associated with gastritis, peptic ulcers, and gastric malignancies. Reliable detection and resistance testing are essential to guide therapy and prevent long-term complications. Culture-based resistance testing is impaired by H. pylori’s fastidious growth. Molecular assays using FFPE gastric biopsies are more practical, but conventional PCR/Sanger sequencing is restricted to a limited set of well-characterized resistance mutations. Samples used for assay validation included both H. pylori positive FFPE samples (n=82) and fresh gastric biopsies that underwent phenotypic testing (n=20). We used the QIAseq xHYB Microbial Hyb&Lib Kit (Qiagen) with custom probes targeting the H. pylori genome and sequenced on the Illumina platform (NextSeq 500) targeting 1-2 million paired-end reads (2x150 bp). Bioinformatic analysis was done in CLC Genomics Workbench (Qiagen). The NGS approach demonstrated robust performance even with fragmented FFPE DNA. It showed excellent concordance with legacy assays for 23S and gyrA resistance mutations. Agreement between the presence of resistance mutations and phenotypic resistance was good for clarithromycin and fluoroquinolones. For other antibiotics, the presence of mutations reported in the literature did not correlate well with phenotypic resistance, requiring further testing. Parallel testing of gastric biopsies by culture and NGS as well as feedback from physicians about prescribed antibiotics and the treatment outcome will allow us to assess the relevance of published mutations and further refine the reported resistance loci. The whole-genome approach allows adaptation to evolving guidelines and further analyses, including the detection of virulence factors and strain profiling.