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Background This study investigated whether the microvascular dysfunction differed between culprit and non‐culprit vessels in patients with acute coronary syndrome who underwent percutaneous coronary intervention. Methods and Results In 115 prospectively recruited patients, after successful percutaneous coronary intervention, culprit and non‐culprit intracoronary hemodynamic measurements were performed and repeated at 6‐month follow‐up. 13N‐ammonia positron emission tomography was performed at 6‐month follow‐up visit to determine absolute myocardial blood flow. The resistance values of each vessel were calculated using the coronary pressure data and the myocardial blood flow values obtained from 13N‐ammonia positron emission tomography data. We compared the measurements between culprit and non‐culprit vessels and assessed changes in microvascular dysfunction during the study period. In 334 vessels (115 culprit and 219 non‐culprit), the culprit vessel group showed a lower fractional flow reserve and coronary flow reserve than the non‐culprit vessel group at baseline and 6‐month follow‐up, respectively. The value of index of microcirculatory resistance was different between the 2 groups in the baseline but not at 6‐month follow‐up. The microvascular resistance at rest and hyperemic microvascular resistance were not different between the 2 groups, but resistance to stenosis was higher in the culprit vessel group, under both resting and hyperemic status (P=0.02 and P