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Ocular coloboma (OC) is a failure of complete optic fissure closure during embryonic development and presents as a tissue defect along the proximal–distal axis of the ventral eye. It is classed as part of the clinical spectrum of structural eye malformations with microphthalmia and anophthalmia, collectively abbreviated to MAC. Despite deliberate attempts to identify causative variants in MAC, many patients remain without a genetic diagnosis. To reveal potential candidate genes, we utilised transcriptomes experimentally generated from embryonic eye tissues derived from humans, mice, zebrafish, and chicken at stages coincident with optic fissure closure. Our in-silico analyses found 10 genes with optic fissure-specific enriched expression: <i>ALDH1A3</i>, <i>BMPR1B</i>, <i>EMX2</i>, <i>EPHB3</i>, <i>NID1</i>, <i>NTN1</i>, <i>PAX2</i>, <i>SMOC1</i>, <i>TENM3</i>, and <i>VAX1</i>. In situ hybridization revealed that all 10 genes were broadly expressed ventrally in the developing eye but that only <i>PAX2</i> and <i>NTN1</i> were expressed in cells at the edges of the optic fissure margin. Of these conserved optic fissure genes, <i>EMX2</i>, <i>NID1</i>, and <i>EPHB3</i> have not previously been associated with human MAC cases. Targeted genetic manipulation in zebrafish embryos using CRISPR/Cas9 caused the developmental MAC phenotype for <i>emx2</i> and <i>ephb3</i>. We analysed available whole genome sequencing datasets from MAC patients and identified a range of variants with plausible causality. In combination, our data suggest that expression of genes involved in ventral eye development is conserved across a range of vertebrate species and that <i>EMX2</i>, <i>NID1</i>, and <i>EPHB3</i> are candidate loci that warrant further functional analysis in the context of MAC and should be considered for sequencing in cohorts of patients with structural eye malformations.