〔Abstract〕 Objective To explore the application value of using next-generation sequencing technology (NGS) to screen the thalassemia in pregnant women. Methods A total of 1036 pregnant women were randomly selected from the obstetrics clinic of Shenzhen Second People's Hospital from November 2018 to January 2019. All pregnant women underwent blood routine and hemoglobin electrophoresis screening. Pregnant women with positive blood routine and hemoglobin electrophoresis screening were further subjected to routine thalassemia gene detection [Gap polymerase chain reaction (GAP-PCR) technology combined with membrane reverse dot hybridization (RBD) technology], and NGS thalassemia gene detection was performed in all 1036 pregnant women. To investigate the carrying status of thalassemia gene in pregnant women in Shenzhen area, and compare the screening effect of thalassemia gene carriers and the additional detection of NGS under different screening modes. Results (1) The overall carrying rate of thalassemia gene was 8.98% in pregnant women, among which the carrying rate of α-thalassemia was 7.05%. β-thalassemia was 1.54%. and the carrying rate of α-thalassemia combined β-thalassemia was 0.39%. A total of 15 thalassemia genotypes were detected in the population, including 1 case of alpha thalassemia with Hb Phnom Penh (HBA1:c.354_355insATC) and 2 cases of beta thalassemia with -50 (G > A). (2) Among the modes of screening thalassemia with different hematological indicators,the combined screening of routine blood and hemoglobin electrophoresis had the highest sensitivity, but there was still at least 12.50% missed diagnosis rate and a large number of false positives. (3) In this study population, NGS results were completely consistent with traditional tertiary screening, and compared with conventional thalassemia genetic testing, an additional 3 cases of unusual types and 4 cases of rare β-globin gene point mutations of unknown significance were detected. Conclusion One-time NGS screening significantly reduces the rate of missed diagnosis in thalassemia gene carriers during the prenatal period, and provides additional detection of rare types and unknown genetic variants.