The role of psychosocial and physical factors in the development of musculoskeletal pain (MSP) has now been clearly demonstrated. However, it is unclear whether these factors contribute to specific regional MSP or to multisite pain. The main goal of this study was to assess the impact of work-related factors according to gender on the development of regional and multisite MSP. A total of 12,591 subjects (65% men and 35% women) who were born in 1938, 1943, 1948, and 1953 and were participating in a French longitudinal prospective epidemiological survey (ESTEV) in 1990 to 1995 were eligible. Personal factors and work exposure were assessed by self-administered questionnaires. Statistical associations between chronic MSP (regional body site or multisite), personal factors, and occupational factors were analyzed using logistic regression modeling. The incidence of regional MSP and multisite pain in 1995 were, respectively, 17% and 25.6%. For women, highly repetitive movements predicted neck/shoulder pain; posture and vibrations predicted arm and low back pain; and effort with tools predicted arm pain. For men, forceful effort and vibrations predicted neck/shoulder pain; posture and forceful effort predicted lower limb and low back pain; and forceful effort and effort with tools predicted arm pain. Physical constraints (ie, forceful effort or vibrations) were associated with multisite pain in both genders. Only for women, psychological factors were risk factors predictive of upper limb pain and in 3 or 4 painful anatomical sites. These results support the hypothesis that some physical and psychological work-related factors are predictive of regional or multisite MSP but differ according to gender. Gender differences and risk factors for work-related musculoskeletal pain should be also taken into account to more effectively target preventive measures.