To investigate the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and plasma concentrations of total homocysteine (tHcy) in Pakistani patients with primary open angle glaucoma (POAG) and primary closed angle glaucoma (PCAG).This was a prospective case-control study. A total of 295 patients (173 POAG, 122 PCAG) and 143 age- and sex-matched controls were subdivided into two ethnic groups, Punjabis (Punjab province, central Pakistan) and Pathans (North-West Frontier Province, northern Pakistan). Genotypes of the MTHFR C677T and A1298C polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). An enzyme-linked immunosorbent assay was used to determine the total serum homocysteine (tHcy) levels. Associations were determined by logistic regression analysis.Frequency distributions of genotypes and combined genotypes as well as homocysteine levels were obtained. The overall distribution of the C677T genotype was found to be significantly associated with PCAG (CC 69%, CT 21%, TT 10%; p=0.001, chi(2)=12.6), but not with POAG (CC 71%, CT 28%, TT 1%; p=0.98, chi(2)=0.02) as compared to the controls (CC 71%, CT 29%, TT 1%). The Pathan cohorts revealed no association with the disease; however, the Punjabis demonstrated a significant association with PCAG (CC 75%, CT 11%, TT 13%; p<0.001, chi(2)=17.2). PCAG in the Punjabi subjects was also significantly associated with the A1298C polymorphism (AA 43%, AC 54%, CC 3%; p<0.001, chi(2)=33.9) as compared to the controls. Combined genotype data showed no association with POAG; however, a significant association with all combined genotypes was observed in the overall PCAG subjects (p<0.05, chi(2)=20.1). This difference was particularly apparent in the TTAA and TTAC combinations that were completely absent in the control groups (p<0.05. chi(2)=49.6). Mean serum tHcy levels were found to be significantly increased in the POAG (15.2+/-1.28 micromol/l, p<0.001) and PCAG (20.8+/-4.8 micromol/l) groups as compared to the controls (10.0+/-0.97 micromol/l). The tHcy levels in the TT and AC genotype were significantly elevated in the PCAG group (67+/-12.39 micromol/l, p<0.001; 23+/-5.94 micromol/l, p=0.027) as compared to the controls.The TT and AC genotypes of MTHFR C677T and A1298C polymorphisms and the combined genotype TTAC were associated with PCAG in Punjabi subjects of Pakistani origin and correlated with the high serum tHcy levels seen in these patients.