Background: High concentrations of homocysteine are considered a risk factor for developing atherosclerosis and coronary artery disease. The aim of this study was to assess the concentrations of homocysteine in subjects with chronic kidney disease (CKD). Methods: Data were collected from medical records of individuals examined at a screening center in Israel between the years 2000–2014. Cross sectional analysis was carried out on 17,010 subjects; 67% were men. Results: Significant differences were observed between four quartiles of homocysteine concentrations and estimated glomerular filtration rate (eGFR)—the higher the homocysteine concentration, the lower the eGFR (p < 0.0001). In subjects with CKD, homocysteine plasma levels were correlated with the stage of renal impairment. Mean (SD) homocysteine concentrations in subjects with eGFR < 60 mL/min per 1.73 m2 compared to subjects with eGFR ≥ 60 mL/min per 1.73 m2 were: 16.3 (5.9) vs. 11.5 (5.5) μmol/L respectively. These findings remained significant after adjustment for age, smoking status, body mass index, hypertension and diabetes mellitus (p < 0.0001). Compared to subjects with homocysteine concentrations less than 15 μmol/L, those with homocysteine concentrations equal and above 15 μmol/L, had a significantly higher odds ratio (95% CI) of having an eGFR < 60 mL/min per 1.73 m2; non adjusted model, 8.30 (6.17–11.16); adjusted model for age smoking status, body mass index, hypertension and diabetes mellitus, 7.43 (5.41–10.21). Conclusion: Plasma homocysteine concentrations are higher in subjects with CKD. This may contribute to an increased risk for developing atherosclerosis and coronary artery disease in these patients.