Congenital eye malformations, such as the microphthalmia-anophthalmia-coloboma spectrum (MAC), anterior segment dysgenesis (ASD), congenital glaucoma (PCG) and congenital cataracts (CC) can cause severe visual impairment from early childhood. Due to phenotypic variability and heterogeneity, with known causative variants in more than 100 genes, establishing a diagnosis in affected patients is often difficult. The aim of this study was to further identify the genetic causes of congenital eye malformations on the base of Next Generation Sequencing (NGS) data and to compare clinical strategies for the diagnosis of these diseases by means of NGS. The aim of this study was to further identify the genetic causes of congenital eye malformations on the base of Next Generation Sequencing (NGS) data and to compare clinical strategies for the diagnosis of these diseases by means of NGS. In detail the following points were analyzed: (1) Workup of known causes of congenital eye malformations and diagnostic yield of different gene panels: Comparison of a phenotype-spanning multigenepanel (MGP) and exome analysis with phenotype-specific primary diagnostic tests (PD) used in clinical routine until now. (2) Genotype-phenotype correlations in congenital eye malformations: Detailed definition and expansion of the possible clinical spectrum of certain genetic variations. (3) Identification of unknown causes of congenital eye malformations: Search for variants in potential new candidate genes and in genes already included in routine diagnostic testing but not yet specifically associated with eye malformations. Patients with MAC, ASD, PCG or CC, who had already undergone primary genetic diagnostic testing (PD) by analyzing single genes or small phenotype-specific panels based on NGS, were included. If PD did not yield results, a stepwise analysis of an eye specific multigene-panel (MGP) containing 186 genes and in the next step exome analysis followed. The analysis ensued in standardized order using the Human Phenotype Ontology (HPO) terminology and the variant classification system the American College of Medical Genetics (ACMG). 8 Out of 40 included patients PD, MGP and exome yielded causative variants in 17 cases (42,5%) in a total of 12 genes (MAB21L2, BCOR, NHS, MFRP, PRSS56, FOXC1, PITX2, CYP1B1, ADAMTS18, COL4A1, PAX6, GCNT2) and relevant variants of unknown significance (VUS) in 6 more cases (15%) in a total of 5 genes (POGZ, COL18A1, FAT1, CRYBB1, MFRP). In 17 cases (42,5%) sequential analysis of PD, MGP and exome yielded no causative variant or phenotype-specific VUS. Primary diagnostic testing revealed causative variants in 6 cases out of all analyzed patients (n=40) across alle phenotypic groups in the respective gene panels, which corresponds to a diagnostic yield of 15%. The eye specific MGP (n=34) containing 186 genes revealed causative variants in 10 additional cases, which corresponds to an isolated diagnostic yield of 29% for this panel and a cumulative diagnostic yield including PD of 40%. Of notice, all genes from the different PD gene panels were also contained in the MGP. Exome analysis (n=24) revealed a causative variant in on more case, corresponding to an isolated diagnostic yield of 4% and a cumulative diagnostic yield for PD, MGP and exome of 43%.