Objective: Reliable data on prevalence of rare differences of sex development (DSD) are lacking. We aimed to estimate population-based prevalence of DSD in Switzerland. Design: Retrospective population-based study including individuals with DSD according to Chicago Consensus, born in Switzerland from 2000-2019. Methods: Endocrine care centers in all ten Swiss Children's Hospitals and eight private endocrine practices collected DSD data through the I-DSD registry or case report forms. We calculated prevalence for DSD diagnostic groups and analyzed time trends in prevalence. Results: Over the 20-year study period, we identified 561 individuals with DSD. Almost half (n=266, 47%) had sex chromosome DSD, 177 (32%) had 46,XY DSD and 118 (21%) had 46, XX DSD. Causes for 46,XY DSD were disturbed androgen synthesis or action (37/177, 21%), atypical gonadal development (28/177, 16%), or other causes (112/177, 63%). Causes for 46,XX DSD were androgen excess (99/118, 84%), atypical gonadal development (8/118, 7%), or other causes (11/118, 9%). On average, 28 new cases were born with DSD annually. Prevalence was 17 for sex chromosome DSD, 12 for 46,XY DSD and 8 for 46,XX DSD per 100'000 live births and year. One per 7'500 newborn girls had 46,XX congenital adrenal hypoplasia (CAH). Conclusion: Prevalence of sex chromosome DSD was lower than expected because of underreporting due to late diagnosis. Prevalence of 46,XX CAH is similar to newborn screening data, suggesting good completeness of cases. For complex DSD cases, we expect complete coverage. This study provides a valuable resource for policymaking and (inter)national research on DSD.