Natural variation in the coding region of the melanocortin-1 receptor (MC1R) gene is associated with constitutive pigmentation phenotypes and development of melanoma and nonmelanoma skin cancers. We investigated the effect of MC1R variants on melanoma using a large, international population-based study design with complete determination of all MC1R coding region variants. Direct sequencing was completed for 2,202 subjects with a single primary melanoma (controls) and 1,099 subjects with second or higher-order primary melanomas (cases) from Australia, the United States, Canada, and Italy. We observed 85 different MC1R variants, 10 of which occurred at a frequency >1%. Compared with controls, cases were more likely to carry two previously identified red hair ("R") variants [D84E, R151C, R160W, and D294H; odds ratio (OR), 1.6; 95% confidence interval (95% CI), 1.1-2.2]. This effect was similar among individuals carrying one R variant and one r variant (defined as any non-R MC1R variant; OR, 1.6; 95% CI, 1.3-2.2) and among those carrying only one R variant (OR, 1.5; 95% CI, 1.1-1.9). There was no statistically significant association among those carrying only one or two r variants. Effects were similar across geographic regions and categories of pigmentation characteristics or number of moles. Our results confirm that MC1R is a low-penetrance susceptibility locus for melanoma, show that pigmentation characteristics may not modify the relationship of MC1R variants and melanoma risk, and suggest that associations may be smaller than previously reported in part due to the study design.