Abstract: Piwi-interacting RNA (piRNA) plays an important role in the gonadal development and maintenance of Teleostei. In this study, piRNA libraries derived from the adult gonads of Japanese flounder (Paralichthys olivaceus) were generated using next-generation sequencing technology. Using zebrafish piRNAs as a reference, 5 865 unique candidate piRNAs were identified; 289 candidate piRNA clusters (PRCs) were generated from the above piRNAs. Among the isolated candidate PRCs, a total of 38 ovary-specific, 45 ovary-bias, 24 testis-specific, and 131 testis-bias PRCs were found. The relative expression levels of seven PRCs were validated through quantitative reverse transcription-polymerase chain reaction. The results of this study will help facilitate exploration of the development and maintenance of the phenotypic sex mechanism in P. olivaceus.