Disorders of sex development (DSD) are a collection of rare congenital conditions with diverse features and pathophysiology. Patients usually present at birth with atypical genitalia or with delayed puberty in adolescence. Biochemical and cytogenetic investigations may provide guidance on the underlying cause, however molecular genetic analysis is usually required to provide a definitive diagnosis and allows for personalised management of the patient. The current diagnostic service for the Scottish population in the West of Scotland Genetics Service detects pathogenic variants in ~10% of cases of XY DSD. Patient samples may also be sent to laboratories in England for further investigations which not only results in additional cost to the National Health Service in Scotland, but also delays treatment and increases anxiety and waiting times for patients and their families. The development of next generation sequencing (NGS) allows multiple genes to be investigated simultaneously at reduced cost and time compared with current methods. A targeted custom SureSelect hybridisation comprehensive gene panel has been designed and validated on the Illumina MiSeq NGS platform. Data analysis was performed using the commercially available software CLC Genomics Workbench and VarSeq. Variant interpretation was conducted using a combination of in silico tools and a specialist multidisciplinary Diagnostic Board with input from endocrinology, clinical and molecular genetics and steroid biochemistry. The validity and clinical utility of this extended gene panel in the diagnostic laboratory has been assessed and will be presented alongside initial results from patients with no previous molecular diagnosis.