Background: Folate and cobalamin are responsible for healthy growth. However, the B-vitamin and homocysteine status of adolescents is not well known. The aim was to assess the status of folate, cobalamin, and homocysteine in healthy Spanish adolescents. Methods: Serum cobalamin, serum folate, homocysteine, methylenetetrahydrofolate reductase 677C>T variant, BMI, smoking habits, and Tanner stage were determined according to gender in 165 adolescents (84 females, 81 males; 13–18.5 years) using the Student’s t test, Mann-Whitney U test and χ2 test, respectively. Interactions between socioeconomic status, age group, methylenetetrahydrofolate reductase polymorphism, BMI, smoking habits, Tanner stage, and vitamin status, respectively, were examined by ANOVA or Kruskal-Wallis H test (p < 0.05). Results: Boys had markedly higher homocysteine (males 8.92 (5.51–22.94) µmol/l; females 7.91 (5.09–13.86) µmol/l), whereas girls showed higher serum cobalamin concentrations (males 540.00 (268.00–946.47) pmol/l; females 594.82 (280.63–1,559.64) pmol/l). Data are shown as medians and the 2.5th to 97.5th percentiles in parentheses. Adolescents with the homozygous variant of methylenetetrahydrofolate reductase displayed significantly higher homocysteine and lower serum folate: normal 5.73 (3.09–10.73) ng/ml serum folate, 7.57 (4.94–12.94) µmol/l homocysteine; homozygous 4.10 (2.75–7.88) ng/ml serum folate, 10.83 (7.00–22.82) µmol/l homocysteine. Conclusion: The present study provides data on the folate, cobalamin, and homocysteine status of Spanish adolescents. To assure a better assessment, revision of references for adolescents is still needed.