The aim of this survey was to study if diet composition could have an impact on plasma folate (B9) and vitamin B12 (B12) concentrations of Holstein cows across the USA and Canada. A total of 22 American (cows = 427) and 24 Canadian (cows = 476) herds participated. Cows were divided into 3 categories: 0–55, 56–120 and 121–200 d in milk (DIM) and according to parity (first, second and third and more). Each dietary ingredient was sampled for wet chemistry analysis. Nutrient composition of the diets was used in statistical analyses. Principal component (PC) analysis was performed on nutrient components of the diet to reduce the number of interdependent variables. Three PC explaining 79% of the variation were retained for analysis as independent variables. Proc MIXED of SAS was used to analyze data (log-transformed). Plasma B9 increased from 12.3 to 14.8 ng/mL between 0 and 120 DIM and reached a plateau afterward. It was greater by 10% for multiparous than for primiparous cows (P < 0.01). Between 56 and 200 DIM, plasma B12 was greater by 21% than before 55 DIM (P < 0.01). Plasma B12 increased as parity number increased (First: 172, 95% confidence interval (CI): 161–184, Second: 185, 95% CI: 173–198 and Third and more: 198, 95% CI: 185–211 pg/mL, P < 0.01). Plasma B9 and B12 were respectively less and greater for American cows (B9: 13.6, 95% CI: 12.9–14.4 ng/mL and B12: 206, 95% CI: 192–221 pg/mL) than for Canadian cows (B9: 14.7, 95% CI: 14.0–15.3 ng/mL and B12: 170, 95% CI: 159–182 pg/mL; P ≤ 0.04). Plasma B9 and B12 were associated with the same PC (PC1; P ≤ 0.02). Plasma B9 was negatively related to fiber (ex. neutral detergent fiber) and crude protein, and positively with nonfiber carbohydrate (P = 0.01) of the diet whereas the opposite was observed for plasma B12 (P = 0.02). Dietary fat (PC2), and Co (PC3) were not related to plasma B9 and B12. These models explained 40 and 42% (pseudo-R²) of the variability of plasma B9 and B12, respectively. In summary, both plasma B9 and B12 concentrations were affected by parity, DIM, and nutrient components of the ration, mainly related to fiber and carbohydrate.