Disorders/Differences of sex development (DSD) represent a major paediatric concern and clinical management of these conditions is often difficult. Uncertainty about a child’s gender can be traumatic and may carry profound psychological and reproductive consequences. Providing a genetic diagnosis for patients with a DSD and their families can serve multiple purposes: naming the underlying cause contributes to acceptance, reduces stigma, and provides crucial guidance for clinical management, including information on the malignancy risks associated with some types of DSD. It is also integral to genetic counseling and family planning. To improve diagnosis rates in DSD we developed a massively parallel sequencing targeted DSD gene panel of 64 known diagnostic DSD genes and 1000 candidate genes. Analysis of DNA from the largest reported international cohort of patients with DSD (327 patients) identified a likely genetic diagnosis in 43% of patients, a significant improvement on the 13% previously achieved in a clinical setting. We found variants in a total of 28 diagnostic genes, with 93 previously unreported variants. Functional testing of many of these has led to improved curation and reporting. This DSD gene panel has now been implemented as a diagnostic test at the Victorian Clinical Genetics Laboratories, Melbourne. Our research focus has shifted to identifying and testing novel DSD genes in the large cohort of patients that we not diagnosed by the panel. Innovative human stem cell technologies and animal models including Drosophila, are being employed to characterize the role of these novel genes in sex development. This includes the gene SART3, in which homozygous variants were identified in patients with sex reversal and intellectual disability. Identification and validation of novel genes such as SART3 will further increase diagnosis rates and improve the health and wellbeing of patients with DSD and their families.