1,3-Butadiene is a known carcinogen primarily targeting lymphoid tissues, lung, and liver. Cytochrome P450 activates butadiene to epoxides which form covalent DNA adducts that are thought to be a key mechanistic event in cancer. Previous studies suggested that inter-species, -tissue, and -individual susceptibility to adverse health effects of butadiene exposure may be due to differences in metabolism and other mechanisms. In this study, we aimed to examine the extent of inter-individual and inter-species variability in the urinary N7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) DNA adduct, a well-known biomarker of exposure to butadiene. For a population variability study in mice, we used the collaborative cross model. Female and male mice from five strains were exposed to filtered air or butadiene (590 ppm, 6 h/day, 5 days/week for 2 weeks) by inhalation. Urine samples were collected, and the metabolic activation of butadiene by DNA-reactive species was quantified as urinary EB-GII adducts. We quantified the degree of EB-GII variation across mouse strains and sexes; then, we compared this variation with the data from rats (exposed to 62.5 or 200 ppm butadiene) and humans (0.004-2.2 ppm butadiene). We show that sex and strain are significant contributors to the variability in urinary EB-GII levels in mice. In addition, we find that the degree of variability in urinary EB-GII in collaborative cross mice, when expressed as an uncertainty factor for the inter-individual variability (UFH), is relatively modest (≤threefold) possibly due to metabolic saturation. By contrast, the variability in urinary EB-GII (adjusted for exposure) observed in humans, while larger than the default value of 10-fold, is largely consistent with UFH estimates for other chemicals based on human data for non-cancer endpoints. Overall, these data demonstrate that urinary EB-GII levels, particularly from human studies, may be useful for quantitative characterization of human variability in cancer risks to butadiene.