Marker-assisted selection (MAS) is fundamental for plant breeding programs, as it can identify desirable seedlings at a young stage and reduce the cost, time and space needed for plant maintenance, especially for perennial crops. To facilitate the process of genotyping, which is time consuming and laborious, we developed a simplified amplicon sequencing (simplified AmpSeq) library construction method for next-generation sequencing that can be applied to MAS in breeding programs. The method is based on one-step PCR with a mixture of two primer sets: the first consisting of tailed target primers, the second of primers that contain flow-cell binding sites, indexes and tail sequences complementary to those in the first set. To demonstrate the process of MAS using s implified AmpSeq, we created databases of genotypes for important traits by using cultivar collections including triploid cultivars and segregating seedlings of Japanese pear (Pyrus pyrifoliaNakai), Japanese chestnut (Castanea crenataSieb. et Zucc.) and apple (Malus domesticaBorkh.). Simplified AmpSeq has the advantages of high repeatability, ability to estimate allele number in polyploid species and semi-automatic evaluation using target allele frequencies. Because this method provides high flexibility for designing primer sets and targeting any variant, it will be useful for plant breeding programs.