<p>Increasing antibiotic resistance in <i>Helicobacter pylori</i> (<i>H. pylori</i>) is undermining empirical eradication therapies. Non-invasive methods for resistance profiling are urgently needed to guide precision treatment. This study aimed to compare the performance of paired gastric juice and stool samples against gastric mucosa for the detection of <i>H. pylori</i> infection and genotypic resistance to clarithromycin and levofloxacin. In this prospective study, patients with <i>H. pylori</i> infection confirmed through positive ¹³C/¹⁴C-urea breath test and histology (Warthin-Starry staining) provided matched gastric mucosal biopsies, gastric juice, and stool samples. Genotypic resistance to clarithromycin and levofloxacin was assessed via commercial quantitative PCR. Performance of gastric juice and stool samples were compared against gastric mucosa as the reference standard using sensitivity, specificity, predictive, and Cohen’s kappa values. Among 218 patients with confirmed <i>H. pylori</i> infection, gastric mucosa identified clarithromycin resistance in 56.9% and levofloxacin resistance in 33.0%. For detecting infection, gastric juice achieved higher sensitivity (97.6% vs. 91.3%, <i>P</i> = 0.003) and specificity (50.0% vs. 30.0%), with stronger reference agreement (kappa = 0.476 vs. kappa = 0.025). In resistance genes profiling, gastric juice exhibited superior sensitivity for both clarithromycin (87.1% vs. 72.6%, <i>P</i> = 0.001) and levofloxacin (70.8% vs. 61.1%, <i>P</i> = 0.049), while maintaining high specificity (82.9% and 92.3%, respectively). Agreement with gastric mucosa was substantially higher for gastric juice (clarithromycin: kappa = 0.691; levofloxacin: kappa = 0.656) than for stool (kappa = 0.504 and kappa = 0.598, <i>P</i> &lt; 0.05 for clarithromycin). Gastric juice also provided higher negative predictive values. Gastric juice shows better diagnostic performance and concordance with mucosal-based resistance genes profiling compared to stool. Furthermore, gastric juice sampling during endoscopy provides a reliable minimally invasive source for resistance profiling, which can serve as a high-quality alternative or backup to mucosal biopsies without the need for repeated invasive procedures.</p><p><b>Registration</b>: This clinical trial was registered at the Chinese Clinical Trial Registry (ChiCTR2400084465).</p>