Objective: This study was conducted to assess whether serum homocysteine concentration was associated with the severity of primary chronic venous disease. Design: Cross-sectional study. Methods: A total of 282 primary chronic venous disease patients were enrolled from outpatient vascular services. The severity of venous disease was graded using the Clinical Etiology Anatomy Pathophysiology classification system. The association of serum homocysteine concentration with advanced primary chronic venous disease (C4-6) was assessed using the Mann Whitney U test and logistic regression analysis. Results: Median (interquartile range) serum homocysteine concentrations were 9.10 µM (7.55–10.75) and 10.40 µM (8.85–13.10) in patients with primary chronic venous disease classified by C1-3 (n = 209) and C4-6 (n = 73) grades, respectively, p < 0.001. Serum homocysteine concentration was positively associated with clinical grade 4–6 after adjusting for other risk factors including age, diabetes, male sex, hypertension, recurrent varicose veins and stroke. Patients with serum homocysteine in the third (odds ratio, 2.76, 95% confidence interval, 1.01–7.54) and fourth (odds ratio 3.29, 95% confidence interval 1.15–9.43) quartiles were more likely to have grade C4-6 chronic venous disease than subjects with serum homocysteine in the first quartile. Conclusions: Serum homocysteine is positively associated with the severity of primary chronic venous disease and therefore could play a role in promoting chronic venous disease complications.