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This study investigates cross-sectional and longitudinal associations between the diversity of leisure-time sport activities and the frequencies of low back pain (LBP) and neck-shoulder region pain (NSP) in twins, including a cross-sectional within-pair design to adjust for potential familial confounding.Finnish twins born in 1975–79 (FinnTwin16 study) reported participation in leisure-time sport activities at the mean ages of 17 (1992–96) (n = 5096, 54% females) and 34 years (2010−12) (n = 3731, 57% females). Diversity assessed as the number of sport activities was categorized as 1, 2, 3, 4, and ≥ 5, excluding inactive individuals. The frequencies of LBP (n = 3201) and NSP (n = 3207), reported at age 34, were categorized as never/seldom, monthly, or weekly pain. Cross-sectional and longitudinal individual-based associations between the number of sport activities and the frequency of LBP and NSP were investigated with multinomial logistic regression analyses, adjusting for multiple confounders. Cross-sectionally, participation in ≥5 sport activities, compared to 1 sport, was associated with significantly less weekly LBP (OR = 0.63, 95%CI = 0.43–0.90), but not with NSP. Longitudinally, participation in several sport activities in adolescence had no significant association with LBP or NSP in adulthood. Cross-sectional within-pair analyses were conducted among twin pairs discordant for LBP (n = 507) and NSP (n = 579). The associations between monozygotic and dizygotic twin pairs were similar in LBP-discordant pairs but differed within NSP-discordant pairs.Participation in ≥5 sport activities in adulthood may be associated with less weekly LBP, but not with monthly LBP or the frequency of NSP. However, within-pair analyses for NSP suggest confounding due to shared familial factors. Keywords: Musculoskeletal pain, Back pain, Multisport, Exercise, Cohort study, Twin study, Behavioral epidemiology