<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Eradication treatment for <jats:italic>Helicobacter pylori</jats:italic> gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese <jats:italic>H. pylori</jats:italic> strains for antimicrobial susceptibility testing.</jats:p></jats:sec><jats:sec><jats:title>Method</jats:title><jats:p>A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Three <jats:italic>H. pylori</jats:italic> strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for <jats:italic>Helicobacter</jats:italic> Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella‐based horse‐serum‐containing agar medium were as follows: JSHR3 (CLR 16 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), JSHR6 (CLR 0.016 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), and JSHR31 (CLR 16 μg/ml, AMX 1 μg/ml and MNZ 64 μg/ml).</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>A reference panel of <jats:italic>H. pylori</jats:italic> JSHR strains was established. The panel consisted of JSHR6, which was antibiotic‐susceptible, JSHR3, which was CLR‐resistant, and JSHR31, which was multi‐resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.</jats:p></jats:sec>