Studies using genome-wide SNPs have become commonplace in genetics and genomics due to advances in high-throughput sequencing technologies. Since the numbers of required SNPs and samples vary depending on each research goal, genotyping technologies with high flexibility in the number of SNPs/samples and high repeatability have been investigated in a wide range of research fields. To achieve this, we used the ultrahigh-multiplexed amplicon sequencing, namely Ion AmpliSeq technology, for constructing reduced representation library and genotyping with NGS platform in cultured fugu, Takifugu rubripes. By using highly multiplexed primer pool, approximately 3,000 SNPs distributed across the whole genome were efficiently and repeatably amplified. To validate and demonstrate this method for aquaculture genomics, we genotyped 652 cultured fugu and thus the genotyping data were used for population structure analysis and genome-wide association study.