Milk is an excellent source of vitamin B₁₂ for humans. Previous studies showed that its concentration is variable among herds. The purpose of this study was to identify diet characteristics affecting milk vitamin B₁₂ concentration (B₁₂) and to verify if maximizing milk B₁₂ could be achieved without affecting income over feed cost (IOFC). A total of 4,440 Holstein cows located in 100 herds participated in the study. One milk sample per cow was collected during the morning milking and analyzed for B₁₂. A sample of each feed ingredient given to the cows within a herd was collected, analyzed by wet chemistry and then nutrient composition of the total mixed ration was calculated. Cost of each ingredient was recorded. Milk B12 was averaged by herd taking into account individual milk production. Proc CLUSTER of SAS was used to divide herds in groups according to milk B₁₂ and IOFC; 4 groups were identified: (1) High B₁₂ (4.4 ng/g) + high IOFC ($18.65 CAN/cow/d); (2) Low B₁₂ (3.9 ng/g) + low IOFC ($12.57 CAN/cow/d); (3) Low B₁₂ (3.5 ng/g) + high IOFC ($16.43 CAN/cow/d) and; (4) High B₁₂ (5.3 ng/g) + low IOFC ($14.55 CAN/cow/d). Proc GLIMMIX of SAS and orthogonal contrasts (1 vs. 4; 1 vs. 3 and; 1+4 vs. 2+3) were used to analyze data. On a herd basis, milk B₁₂ averaged 4.1 ng/g (min-max: 2.6–5.9 ng/g). When comparing herds with high B₁₂, IOFC was higher when diets contained more non-fiber carbohydrates (+3.3% dry matter [DM]; NFC) and less acid (−2.5%; ADF) and neutral detergent fibers (−3.8% DM; NDF; P < 0.02). In higher IOFC herds, B₁₂ of milk was higher in herds in which NFC, ADF and NDF averaged 42.4, 20.6, 35.3% compared with 38.9, 22.9, 39.4% on a DM basis for herds with lower B₁₂ (P < 0.0002). Moreover, diets of herds with higher B₁₂ contained 7.4 and 6.6% DM less hay and physically effective NDF, respectively (P < 0.02). In line with previous results, regardless of IOFC, high B₁₂ herds were characterized with diets containing more NFC (+2.7% DM), less ADF and NDF (−1.7 and −3.2% DM, respectively), more grass silage (+11.2% DM) and less hay (−8.9% DM; P < 0.04) than low B₁₂ herds; maximizing milk B12 can be achieved without decreasing IOFC.