Objective To investigate the clinical application value of chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) in fetuses with increased nuchal translucency (NT). Methods From January 2020 to April 2022, clinical data of 1013 fetuses were collected who underwent invasive prenatal diagnosis due to NT thickening [defined as NT ≥95th centile for the crown-rump length (CRL)] in the Third Affiliated Hospital of Zhengzhou University. All fetuses were undergone CMA detection, 49 fetuses with negative CMA results underwent further prenatal WES. According to NT value, fetuses were divided into the following four groups: <3.5 mm group (529 cases, 21 cases underwent WES), 3.5-4.5 mm group (273 cases, 8 cases underwent WES), 4.5-5.5 mm group (98 cases, 7 cases underwent WES), and ≥5.5 mm group (113 cases, 13 cases underwent WES). According to the results of ultrasound examination, all fetuses were divided into structural malformation group (88 cases, 23 cases underwent WES) and isolated increased NT group (925 cases, 26 cases underwent WES). The possible chromosomal anomalies were analyzed by CMA first. Furthermore, 49 cases with increased NT but negative CMA results were investigated by WES, and the outcomes were followed up. Results CMA showed that, among the 1013 cases of NT thickened fetus, 224 cases (22.1%) of causative genetic defects were detected, including 182 (18.0%) cases with chromosomal aneuploidy and 42 (4.1%) cases with pathogenic copy number variation (pCNVs). Among different NT value groups, the positive rate of CMA in NT ≥5.5 mm group was the highest (47.8%). In addition, the positive rate of CMA in fetuses with increased NT and structural malformations was higher than in isolated increased NT (45.5% vs. 19.9%). WES detected monogenic disease in 5 of 49 fetuses (10.2%) with increased NT and negative CMA results, including 3 cases of autosomal dominant disease, 1 case of autosomal recessive hereditary disease and 1 case of X-linked recessive hereditary disease. All the five fetuses had structural malformations, two of them with increased NT <3.5 mm, and only one of them was born alive. Conclusion WES can detect out monogenic disease in fetuses with increased NT combined structural malformations with negative CMA results. Fetuses with increased NT should also be alert for the possibility of monogenic disease even if the NT value less than 3.5 mm.