BACKGROUND: In 2011, a US expert panel recommended universal cholesterol screening for children ages 9 to 11 years. Controversy exists over this recommendation, especially because the most recent systematic review on universal childhood screening was inconclusive. OBJECTIVES: To conduct an updated systematic review on universal cholesterol screening in childhood and effect on health outcomes, clinical management, screening acceptability, and healthcare costs. DATA SOURCES: We searched MedLine, EMBASE, Psychinfo, and the Cochrane Registry of Controlled Trials from October 2005 to January 2016. We added new studies identified to those from the previous systematic review (1966-September 2005). STUDY ELIGIBILITY, PARTICIPANTS, AND INTERVENTIONS: We included controlled trials, pre-post, cohort, survey, and qualitative studies of universal cholesterol screening in children ages 0 to 18 years. STUDY APPRAISAL AND SYNTHESIS METHODS: Two independent reviewers assessed abstracts and full-text studies, extracted data, and ranked quality. Cost data were inflation-adjusted to 2015 dollars. RESULTS: Nine new studies met inclusion criteria, taking the total number of relevant studies to 21. Screening was associated with no change in cholesterol in 1 of 1 study on health outcomes. A positive screen for dyslipidemia was associated with diet and/or exercise changes in 29% to 92% of families in 4 of 4 studies. Adherence with new guidelines for universal screening was low (16%-18%) in 3 of 3 studies. Costs per case of familial hypercholesterolemia detected were $12,500 to $20,300. LIMITATIONS: Included studies were heterogeneous in outcomes. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: Universal cholesterol screening might have small, positive effects on lifestyle change, but the effect on health remains understudied.