AbstractVariants in the CNGB3 gene, encoding the B3‐subunit of the cone photoreceptor cyclic nucleotide gated channel, are a major cause of autosomal recessive achromatopsia, a rare inherited retinal disease. The mutation spectrum of achromatopsia‐associated CNGB3 variants comprises all types of mutations, including those that are straightforward to evaluate in molecular genetic diagnostics, such as frame‐shifting, nonsense, and canonical splice site variants. Additionally, variants have been identified within splice regions outside the conserved ±1,2 splice site dinucleotides, making their potential impact on disease association challenging to interpret. This poses a major hurdle for clinical interpretation of causality between the patient's genotype and the proposed clinical diagnosis, but also for the inclusion of such patients into clinical trials for gene augmentation therapy, for which only patients with confirmed (likely) pathogenic CNGB3 variants are eligible. We here performed comprehensive genetic functional analysis of 21 candidate spliceogenic CNGB3 variants—15 reported and 6 novel variants—by means of in vitro minigene splice assays and cDNA analysis, and characterization of spliceogenic events by subcloning, Sanger‐sequencing, and capillary fragment analysis. For 16 variants, an impact on splicing was confirmed, supporting the reclassification of 86% of variants of uncertain significance as likely pathogenic or pathogenic according to the ACMG/AMP guidelines. This reclassification enables the confirmation of patients’ genotypes, both retrospectively and prospectively. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.