AIM: To conduct a genetic analysis of Han-Chinese patients with isolated congenital ptosis (ICP) and identify the genetic variants related to the condition. METHODS: Sixty-five unrelated patients with ICP were enrolled. Comprehensive clinical examinations, whole exome sequencing (WES), and Sanger sequencing were used to reveal the potential genetic causes. Combined with public and in-house control databases, multiple bioinformatics prediction tools, and conservation analysis, the potential variants were further analyzed. AlphaFold 3, an accurate modelling prediction tool, was utilized to generate three-dimensional structural models of both wild-type and mutated proteins. RESULTS: Three novel heterozygous variants in the zinc finger homeobox 4 gene (ZFHX4), c.5145C>A (p.N1715K), c.10382C>T (p.A3461V), and c.10795G>A (p.A3599T), were identified in three patients, respectively. Bioinformatics analyses suggested that these variants are likely to exert deleterious effects, supporting their potential involvement in the pathogenesis of ptosis. CONCLUSION: The novel heterozygous ZFHX4 variants are identified as disease-associated variants in three patients with ptosis, suggesting that ZFHX4 may be a disease-causing gene for autosomal dominant ICP with incomplete penetrance or a susceptibility gene. These findings expand the variant spectrum of ZFHX4, improve understanding of the pathogenesis of ZFHX4-related ptosis, and may contribute to the genetic counseling and disease management, as well as the development of experimental treatments.