Extensive effort is dedicated to genotyping human, plant and animal populations, to uncover genetic relationships and identify genes that regulate clinical and agricultural traits, among many other uses. With the dramatic increase in DNA sequencing throughput and reduction in cost, direct sequencing of reduced genome representations has emerged as an option for genotyping. Reduced genome representations have been typically generated by restriction enzyme digestion, adaptor ligation, and selective PCR amplification, followed by sequencing. However, in addition to requiring a series of sample-specific enzymatic steps, the approach is restricted by the existing enzymes, limiting the flexibility in marker coverage and density. Here an alternative approach is proposed, that uses a two-step PCR, intercalated by a normalization procedure. Briefly, the first PCR reaction begins with the amplification of regions in the genome with primers containing a specific sequence in the 3’end, followed by degenerate nucleotides, and a tail that is complementary to the primer used in the second step of the PCR reaction. Before the second PCR, digestion with a duplex-specific nuclease is carried out to reduce representation of overly abundant fragments. In the second PCR, linkers that allow sequencing in nextgen sequencers are incorporated. Both normalization and the second PCR reaction are done by multiplexing 48-96 samples, allowing for a single technician to process several hundred samples daily, using standard laboratory equipment. Finally, we demonstrate the method in the detection and segregation analysis of ~10,000 markers in a mapping population of Eucalyptus.