A discrepancy has been identified between numbers of expected and identified patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. Patients homozygous for the frequent c.833T>C (p.I278T) are most often responsive to vitamin B6, and can present with a total-homocysteine (tHcy) <100μM on a normal diet. In Denmark, patients with tHcy <100μM are not routinely sequenced for CBS22Cystathionine beta-synthase mutations. This study investigated the prevalence of CBS mutations and the common methylenetetrahydrofolate reductase (MTHFR) c.677C>T polymorphism in patients with tHcy ≥50μM and the association with clinical manifestations. We studied a cohort of patients with intermediate and severe hyperhomocysteinemia (tHcy ≥50μM) determined between 1996 and 2011. Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR33Methylenetetrahydrofolate reductase c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS2. Of those, two were homozygous for c.833T>C (p.I278T), and four were compound heterozygous for c.833T>C. One c.833T>C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100μM to 50μM. The most prominent clinical presentation among patients with a CBS2 mutation was thrombosis presenting at a median age of 25years. In case of arterial or venous thrombosis without any explanation in individuals below 40years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100μM genotyping for the MTHFR3 c.677TT is relevant, and when tHcy >100μM CBS should be genotyped.