Hypertension and hyperhomocystinemia have a joint effect on the risk of stroke. We aimed to evaluate the relationship between plasma total homocysteine (tHcy) and blood pressure in two independent Chinese populations. Four thousand five hundred and fifty-five participants who underwent health examinations between March 2016 and September 2016 at Peking University First Hospital were enrolled as 'Population 1', and 2689 participants who were admitted to Peking University First Hospital between January 2014 and December 2015 were enrolled as 'Population 2'. None of the study participants were taking antihypertensive medication or vitamins, or had cardio-cerebrovascular disease or chronic kidney disease stages 4 or 5. In Population 1, a 5 μmol/L increase in tHcy was associated with a 0.47 mmHg (95% confidence interval [CI]: 0.23-0.70 mmHg, p < 0.01) increase in systolic blood pressure (SBP) and a 0.14 mmHg (95% CI: -0.02 to 0.30 mmHg, p = 0.08) increase in diastolic blood pressure (DBP). In Population 2, a 5 μmol/L increase in tHcy was associated with a 0.42 mmHg (95% CI: 0.13-0.72 mmHg, p < 0.01) increase in SBP and a 0.29 mmHg (95% CI: 0.09-0.49 mmHg, p < 0.01) increase in DBP. The prevalence of hypertension was significantly higher in Population 1 (by 47%; odds ratio [OR] 1.47, 95% CI: 1.09-1.98, p = 0.01) and in Population 2 (by 55%;OR 1.55, 95% CI: 1.15-2.08, p < 0.01) in participants with tHcy ≥ 15 μmol/l than in those with tHcy < 10 μmol/L. Stratified analysis showed that the association was stronger in women than in men.