BACKGROUND/OBJECTIVES It has been suggested that the association between shift work and chronic disease is mediated by an increase in obesity. However, investigations of the relationship between shift work and obesity reveal mixed findings. Using a recently developed exposure assessment tool, this study examined the association between shift work and obesity among Canadian women from two studies: a cohort of university alumni, and a population-based study. METHODS Self-administered questionnaire data were used from healthy, currently employed females in a population-based study, the Ontario Women's Diet and Health case-control study (n = 1611 controls), and from a subset of a of university alumni from the Canadian Study of Diet, Lifestyle, and Health (n = 1097) cohort study. Overweight was defined as BMI≥25 to <30, and obesity as BMI≥30. Reported occupation was converted to occupational codes and linked to a probability of shift work value derived from Survey of Labour and Income Dynamics data. Regular evenings, nights, or rotating work comprised shift work. Polytomous logistic regression estimated the association between probability of shift work, categorized as near nil, low, medium, and high probability of shift work, on overweight and obesity, controlling for detected confounders. RESULTS In the population-based sample, high probability of shift work was associated with obesity (reference = near nil probability of shift work, OR: 1.88, 95% CI: 1.01-3.51, p = 0.047). In the alumni cohort, no significant association was detected between shift work and overweight or obesity. CONCLUSIONS As these analyses found a positive association between high probability of shift work exposure and obesity in a population-based sample, but not in an alumni cohort, it is suggested that the relationship between shift work and obesity is complex, and may be particularly susceptible to occupational and education-related factors within a given population.