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The aim of the present study was to investigate the association between serum levels of vitamins B12, B6, folic acid and hyperhomocysteinemia with the risk of coronary heart disease (CHD). A case control study was carried out involving 105 newly diagnosed coronary heart disease patient of both gender (age range 25–60 years) and 105 age and sex matched normal healthy individuals at Punjab Institute of Cardiology, Lahore. Fasting blood samples were collected from both cases and controls. Sera were analyzed for folic acid, vitamin B6 and B12 using high performance liquid chromatography (HPLC). Plasma homocysteine was analyzed by fluorescence polarization immunoassay (FPIA) and traditional risk factors were also assessed. Mean plasma homocysteine level in cases (22.33 ± 9.22 μmol/L) was significantly higher (P = 0.0001) than controls (12.5 ± 3.73 μmol/L). Mean serum folic acid concentration, serum vitamin B6 and vitamin B12 were significantly lower in cases than the mean levels in controls (P < 0.05). When compared to controls the data revealed significantly greater deficiency of folic acid, vitamin B6 and B12 in coronary heart disease patients. Statistically significant inverse correlation was observed between folic acid, vitamin B6, B12 and plasma homocysteine concentration (r = −0.463, r = −0.431 and r = −0.294, respectively). Nutritional deficiency of folic acid, vitamin B12 and B6 were common in coronary heart disease patients with an inverse correlation of plasma homocysteine concentration with these vitamins. Substantial deficiency of these vitamins along with hyperhomocysteinemia could be further aggravating the risk of CHD in Pakistani population. [ABSTRACT FROM AUTHOR]