OBJECTIVE Traditional Helicobacter pylori (H. pylori) eradication therapy has been undermined by increasing antimicrobial, especially clarithromycin, resistance. Susceptibility testing in some areas is difficult to achieve or unavailable. We aimed to determine whether gastric biopsy specimens stored at room temperature for rapid urease test (RUT) were suitable for clarithromycin susceptibility testing of H. pylori. METHODS After 30 days of storage at room temperature, DNA was extracted from gastric biopsies present in RUTs (Hpfast). H. pylori status and clarithromycin susceptibility were evaluated using H. pylori-specific polymerase chain reaction (PCR) for ureA, vacA, and allele-specific primer-PCR of the 23S rRNA genes. The PCR results were compared with histology, RUT, and culture results. H. pylori positive was defined as RUT and either culture or histology positive; H. pylori negative as RUT, culture and histology negative. RESULTS Samples from 31 patients were evaluated; 11 were H. pylori positive including 9 by culture; seven of which had allele-specific primer-PCR results from the RUT specimen for the detection of mutations of the 23S rRNA gene. When both tests were available, culture and PCR results were concordant in 7 cases. In 15 of the 20 histology, RUT and culture negative patients, three PCR were negative. In one patient, all of the three tests were positive; and in three only the 23S rRNA was positive and in one only ureA was positive. CONCLUSIONS Gastric biopsy specimens stored in the gel of RUT for 30 days can be used for molecular testing to confirm the diagnosis of H. pylori infection and test for clarithromycin susceptibility.