Abstract We estimated the prevalence of musculoskeletal pain in five anatomical areas and their interference with daily activities (IDA) in a Japanese adult population (n = 3188). A questionnaire survey was conducted among participants in health examinations at three health care centers. On a drawing with predefined body regions, participants marked the regions affected by pain and the regions affected by IDA due to the pain, respectively. Overall prevalence of musculoskeletal pain was 41.4% (M 40.9%, F 42.2%) with a significant increase with age. Neck and shoulder area showed the highest prevalence of pain (20.3%; M 19.6%; F 21.3%), followed by lower back area (19.1%; M 20.1%; F 17.5%), hip and knee area (11.1%; M 9.1%; F 14.3%), elbow, wrist, and hand area (7.4%; M 6.6%; F 8.6%), and ankle and foot area (5.8%; M 5.5%; F 6.3%). Overall percentage of IDA in musculoskeletal pain was 20.5%. After adjustment for age and sex, the ranking of percentage of IDA in the pain was: (1) neck and shoulder area (31.0%), (2) elbow, wrist, and hand area (21.5%), (3) ankle and foot area (18.8%), (4) lower back area (17.9%), and (5) hip and knee area (17.4%). Overall percentage of treatment for musculoskeletal disorders was 6.6% (M 4.7%, F 9.7%), which remained 28.1% (M 21.6%, F 38.0%) even in those who described IDA due to pain. According to our estimates, 42.2 million (41.2%) of Japanese adults might suffer from musculoskeletal pain and 9.1 million (8.8%) might encounter IDA due to the pain. Because of high prevalence and IDA, musculoskeletal pain is one of the health problems to be given high priority in Japan.