Objective: To investigate the value of Whole exome sequencing (WES) in the control analysis of prenatal ultrasound diagnosis of fetal abnormalities (including structural malformations and soft indicators) whose etiology was not clarified by Chromosome microarray analysis (CMA)..METHODS: Eighty-one patients with negative results of invasive prenatal diagnosis (CMA) after ultrasound examination of fetal abnormalities detected at the Maternity Hospital of Nanjing Medical University from January 2022 to January 2024 were selected for WES. Genetic variants were classified according to the American Society for Medical Genetics and Genomics Genetic Variation Classification Criteria and Guidelines. Pathogenic and possibly pathogenic variants were categorized as positive results, and clinical significance unknown, benign, and possibly benign were categorized as negative results. RESULTS: The 81 ultrasound anomalies consisted of 47 monosystemic (58.02%) and 34 multisystemic (41.98%) anomalies.WES detected a total of 14 (17.28%) positives, including 7 each of monosystemic and multisystemic anomalies, while the remaining 67 were negative (82.72%). The most common ultrasound abnormalities in positive fetuses were cardiovascular system abnormalities and skeletal system abnormalities, both in 5 cases (35.71%), followed by urinary system abnormalities in 4 cases (28.57%). In addition, 2 fetuses had combined nuchal translucency (NT) thickening at early stage (14.29%), and multiple abnormalities were found by ultrasound at mid-trimester. CONCLUSION: Fetuses with ultrasound anomalies, especially when combined with cardiovascular, skeletal, urinary anomalies or multisystem anomalies, are recommended to undergo WES testing if CMA testing fails to clarify the etiology, which may identify new potential causative genes.