While various technologies for high-throughput genotyping have been developed for ecological studies, simple methods tolerant to low-quality DNA samples are still limited. In this study, we tested the availability of a random PCR-based genotyping-by-sequencing technology, genotyping by random amplicon sequencing, direct (GRAS-Di). We focused on population genetic analysis of estuarine mangrove fishes, including two resident species, the Amboina cardinalfish (Fibramia amboinensis, Bleeker, 1853) and the Duncker's river garfish (Zenarchopterus dunckeri, Mohr, 1926), and a marine migrant, the blacktail snapper (Lutjanus fulvus, Forster, 1801). Collections were from the Ryukyu Islands, southern Japan. PCR amplicons derived from ~130 individuals were pooled and sequenced in a single lane on a HiSeq2500 platform, and an average of three million reads was obtained per individual. Consensus contigs were assembled for each species and used for genotyping of single nucleotide polymorphisms by mapping trimmed reads onto the contigs. After quality filtering steps, 4,000-9,000 putative single nucleotide polymorphisms were detected for each species. Although DNA fragmentation can diminish genotyping performance when analysed on next-generation sequencing technology, the effect was small. Genetic differentiation and a clear pattern of isolation-by-distance was observed in F. amboinensis and Z. dunckeri by means of principal component analysis, F-ST and the admixture analysis. By contrast, L. fulvus comprised a genetically homogeneous population with directional recent gene flow. These genetic differentiation patterns reflect patterns of estuary use through life history. These results showed the power of GRAS-Di for fine-grained genetic analysis using field samples, including mangrove fishes.