This systematic literature review assessed the global prevalence and birth prevalence of clinically significant forms of alpha- and beta-thalassemia. Embase, MEDLINE, and the Cochrane Library were searched for observational studies published January 1, 2000, to September 21, 2021. Of 2093 unique records identified, 69 studies reported across 70 publications met eligibility criteria, including 6 records identified from bibliography searches. Thalassemia prevalence estimates varied across countries and even within countries. Across 23 population-based studies reporting clinically significant alpha-thalassemia (e.g., hemoglobin H disease and hemoglobin Bart's hydrops fetalis) and/or beta-thalassemia (beta-thalassemia intermedia, major, and/or hemoglobin E/beta-thalassemia), prevalence estimates per 100 000 people ranged from 0.2 in Spain (over 2014-2017) to 27.2 in Greece (2010-2015) for combined beta- plus alpha-thalassemia; from 0.03 in Spain (2014-2017) to 4.5 in Malaysia (2007-2018) for alpha-thalassemia; and from 0.2 in Spain (2014-2017) to 35.7 to 49.6 in Iraq (2003-2018) for beta-thalassemia. Overall, the estimated prevalence of thalassemia followed the predicted pattern of being higher in the Middle East, Asia, and Mediterranean than in Europe or North America. However, population-based prevalence estimates were not found for many countries, and there was heterogeneity in case definitions, diagnostic methodology, type of thalassemia reported, and details on transfusion requirements. Limited population-based birth prevalence data were found. Twenty-seven studies reported thalassemia prevalence from non-population-based samples. Results from such studies likely do not have countrywide generalizability as they tended to be from highly specific groups. To fully understand the global prevalence of thalassemia, up-to-date, population-based epidemiological data are needed for many countries.