Classical aniridia is an eye malformation caused by the loss of one working copy of the PAX6 gene, but a minority of cases remain genetically unsolved. We sequenced the whole genomes of 37 affected families (51 individuals) where PAX6 had previously been sequenced but no causative mutation was found. We found a genetic cause in 22 (60%) families. 19 cases had variants (some never before seen) predicted to disrupt PAX6 or its regulation, highlighting the strong disease-gene link. Two thirds were caused by large structural mutations in the genome, rather than single DNA base changes. Three cases had causative variants in the FOXC1 gene, known to cause a form of aniridia. We conclude that whole genome sequencing proves an effective diagnostic test in most individuals with previously unexplained aniridia. (By Dr. Nikki Hall, https://jmg.bmj.com/content/early/2023/11/30/jmg-2023-109181 )