Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and
post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. To determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, Cynoglossus semilaevis, were identified, and their small RNAs were sequenced and analysed. We used C. semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed ''pseudo-males'' who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring. We identified six signature piRNAs as biomarkers in seminal plasma exosomes from males and pseudo-male C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylation related and transposition-related piRNAs. Their expression profiles were verified by using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudo-males. The signature piRNAs could be exploited as male specific biomarkers in this fish. As far as we know, these are first male specific biomarkers in C. semilaevis.Also, it is the first report that piRNAs are employed as biomarkers in fish.These signatures provide an effective tool to explore sex typing in C. semilaevis and may rovide guidance for future research on the generative mechanism of sex reversed ''pseudo-males'' in C. semilaevis.