Abstract Objectives In vivo hemolysis is associated with thromboembolism. Although an increased Hemolysis Index (HI) can be due to in vitro as well as in vivo hemolysis, both reflects a more fragile erythrocyte population. We therefore hypothesized that HI above upper reference limit would be associated with an increased risk of cardiovascular disease (CVD). Methods We identified persons with two elevated HI (HI+) from blood samples analyzed at a university hospital laboratory from 2012 to 2017. We compared their risk of CVD with the risk in matched comparators with normal HI and from the general population. HI+ persons and comparators were followed from start date (date of the second elevated HI) until the first of the main outcome: CVD, emigration, death, or end of observation time on December 31, 2018. Results In 43,102 unique HI+ persons, the risk of developing CVD was 40% higher compared with the general population and 13% higher compared with the matched blood sample cohort. HI+ was associated with a significantly increased cumulative incidence of both arterial and venous CVD compared with the matched blood sample cohort and the general population (respectively 47 and 14% for arterial CVD; 78 and 24% for venous CVD). Moreover, overall mortality risk was significantly higher in patients with HI+ than in the two comparator groups. Conclusions Elevated HI is associated with increased risk of arterial and venous CVD and with increased mortality. Our findings imply that HI may contribute as a CVD risk biomarker.