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Ayu (Plecoglossus altivelis) is a commercially important common osmeroid fish for freshwater fisheries. But little is known about the sex-determination (SD) mechanisms. Type IIB endonucleases restriction-site associated DNA sequencing (2b-RAD-seq) is a popular reduced representation method and shown to be an efficient and valuable way for identifying the sex-specific DNA fragments and SD systems. In this study, one male and one female were first whole-genome sequenced. The total size of high-quality genome assembly of the male was 434 Mb (14,900 scaffolds) and of the female 424 Mb (15,340 scaffolds). And ten males and ten females were then subjected to 2b-RAD-seq and isolated 124 candidate male-specific tags and no female-specific tags. The candidate male-specific tags were mapped to 14 male scaffolds, nine of which were aligned to the female allelic scaffolds, and the other five belonged to the candidate male-specific scaffolds. Thereinto, scaf162525, scaf184183, scaf192610 encoded 45 candidate genes in all, 9 of which had functional descriptions. Furthermore, the anti-Mullerian hormone type-2 receptor on scaf192610 was tentatively identified as a Y-linked duplicate copy (Amhr2by) of the autosomal counterpart (Amhr2a), demonstrating the potential role in the SD system of ayu. Based on the four selected male-specific loci, four sets of primers were designed to develop the multiplex-PCR assay. Results showed that the four male-specific markers could identify the genetic sex with 100% accuracy in 126 farmed mature ayu of the different source populations. In conclusion, the genome sequences from both sexes combined with 2b-RAD sequencing analyses from other males and females led to the isolation of male-specific scaffolds, supporting an XX/XY SD system in ayu. The multiplex-PCR assay based on male-specific markers can be applied to perform molecular sexing in ayu culture.