Vitamin D and folate are associated with decreased colorectal cancer risk and their association with colorectal cancer prognosis is under investigation. We assessed the levels of plasma 25-hydroxyvitamin D₃ (25(OH)D₃), folate and vitamin B₁₂ in an international pilot study in order to determine variability of these biomarkers based on geographical location. Plasma 25(OH)D₃, folate and vitamin B₁₂ concentrations were measured in 149 invasive, newly diagnosed colorectal cancer cases from Heidelberg (Germany), Seattle (WA, USA), and Tampa (FL, USA) and in ninety-one age- and sex-matched controls. Their associations with potential predictors were assessed using multivariate linear regression analyses. Plasma 25(OH)D₃, folate and vitamin B₁₂ concentrations differed by location. Other predictors were season for 25(OH)D₃ and tumour stage (vitamin B₁₂). Season-corrected average 25(OH)D₃ concentrations were higher in Heidelberg (31·7 ng/ml; range 11·0–83·0 ng/ml) than in Seattle (23·3 ng/ml; range 4·0–80·0 ng/ml) and Tampa (21·1 ng/ml; range 4·6–51·6 ng/ml). In Heidelberg, a strong seasonal variation was observed. Folate (11·1 ng/ml) and vitamin B₁₂ (395 pg/ml) concentrations in Heidelberg were lower than those in Seattle (25·3 ng/ml and 740 pg/ml, respectively) and Tampa (23·8 ng/ml and 522 pg/ml, respectively). Differences in plasma 25(OH)D₃ and folate concentrations between Heidelberg and the US sites were observed, probably reflecting variation in outdoor activities and sun-avoidance behaviour during summer as well as in folic acid fortification and supplement use. Intra-site differences at each study location were greater than between-location variability, suggesting that individual health behaviours play a significant role. Nevertheless, the intra-site differences we observed may be due to chance because of the limited sample size. Our pilot study illustrates the value of an international cohort in studying colorectal cancer prognosis to discern geographical differences in a broad range of exposures.