We developed a PCR-based procedure for constructing reduced representation libraries without RE digestion steps, de novo SNP discovering, and its genotyping with NGS platform. By using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were effectively amplified from a wide variety of genomes without prior genetic information. To validate and demonstrate this method, we sequenced 84 genomic libraries from eight non-model species (a fungus, three plants, three invertebrate and a vertebrate).