BACKGROUND: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of disorders affecting millions worldwide. Despite the widespread adoption of next-generation sequencing (NGS) panels, there remains a critical gap in the genetically diverse and understudied African populations. METHODS: One hundred and thirty-five South African patients affected by various IRDs underwent NGS using a custom-targeted panel sequencing over 100 known genes. The panel was supplemented by in silico screening for a MAK-Alu insertion and screening of seven functionally established deep intronic variants. RESULTS: Through our combined screening strategy, we obtained a probable genetic diagnosis for 56% of the cohort. We identified 83 unique variants in 29 IRD genes underlying the disease, including 16 putative novel variants. Molecular findings prompted recommendations for clinical re-examination in ten patients. Resolution rates varied across clinical classifications and population groups. CONCLUSIONS: This study reports the first use of a targeted NGS panel for IRDs in southern Africa, demonstrating a cost-effective, customisable approach that optimises both diagnostic yield and resource efficiency, making it a valuable tool for IRD molecular characterisation in resource-limited settings. Augmenting the panel by screening for variants relevant to South African patients allowed us to achieve a resolution rate in line with international studies. Our study underscores the importance of investigating diverse populations to bridge disparities in genomic research and improve diagnostic outcomes for underrepresented population groups.