Cardio-embolic ischemic stroke caused by atrial fibrillation is more severe compared with other types of stroke, such as lacunar infarction and atherothrombotic infarction in patients without atrial fibrillation. Therefore, it is important to prevent cardio-embolic ischemic stroke by detecting atrial fibrillation early in at-risk patients and administering appropriate anticoagulation therapy. This prospective observational study aimed to evaluate the effectiveness of opportunistic atrial fibrillation screening at 12 primary clinics in Japan. The study included a 12-month pre-campaign period and a 12-month campaign period. During the campaign period, an awareness campaign was conducted to encourage physicians to be mindful of screening patients aged ≥65 years for atrial fibrillation by checking their pulses and performing subsequent electrocardiography when an irregular pulse was detected. The primary outcome was the proportion of patients with newly diagnosed atrial fibrillation. A sub-analysis focusing on first-time outpatients was performed. There were 9921 and 10,282 patients with no history of atrial fibrillation in the pre-campaign and campaign periods, respectively. In the whole population, the proportion of patients with newly diagnosed atrial fibrillation was 0.9% throughout the pre-campaign and campaign periods. In the sub-analysis limited to first-time outpatients, the detection proportion increased from 1.6% to 1.9% during the campaign period. In terms of age stratification, a large increase in detection was observed, especially among patients aged 65-74 years (detection increased from 0.9% to 1.5%) and ≥85 years (detection increased from 2.9% to 3.3%) during the campaign period. Our findings suggest the feasibility of opportunistic atrial fibrillation screening in routine primary care practice in Japan. Of note, our findings suggest that opportunistic atrial fibrillation screening targeting first-time outpatients may be of clinical value.