The restriction-site associated DNA sequencing (RAD-seq) is a high-throughput sequencing technique developed from the next-generation sequencing (NGS). This method can reduce the representation of the complex genome while mapping thousands of polymorphic markers with or without a reference genome. It has been extensively used for high-density genetic map construction, fine mapping of important genes, genome sequence assembly, population genomic research, as well as phylogenetic research and so on. Here, we introduce the technological principle and development of RAD-seq combined with the sequencing applications in various species. Due to its uniqueness, RAD-seq will have a wide application in genetic analysis of complex genomic research in the future.