We identified 21 novel RVs of nine genes, which extended the genetic spectrum of 46, XY DSD pathogenic variants. Our study showed that 60% of the patients were caused by AR, SRD5A2 or NR5A1 P/LP variants. Therefore, PCR amplification and Sanger sequencing of these three genes could be performed firstly to identify the pathogeny of the patients. For those patients whose pathogenic variants had not been found, whole-exome sequencing could be helpful in determining the etiology. This article is protected by copyright. All rights reserved.