| Summary: | Hainan medaka (<i>Oryzias curvinotus</i>) is distributed in the coastal waters of the South China Sea and is able to adapt to a wide range of salinities. In this study, we characterized <i>O</i><i>. curvinotus</i> in Sanya River (SY-medaka), which lacks <i>dmy</i> (a male sex-determining gene in <i>O. latipes</i> and <i>O. curvinotus</i>). In a comparison of SY-medaka and Gaoqiao medaka (GQ-medaka), the morphological difference between the two populations does not reach the subspecies level and they can be considered two geographic populations of <i>O. curvinotus</i>. A mitochondrial cytochrome oxidase subunit I (CoI) sequence alignment showed that the sequence identities between SY-medaka and other geographic populations of <i>O. curvinotus</i> are as high as 95%. A phylogenetic analysis of the mitochondrial genome also indicated that SY-medaka belongs to <i>O. curvinotus</i>. Molecular marker-based genetic sex assays and whole genome re-sequencing showed that SY-medaka does not contain <i>dmy</i>. Further, in RNA-Seq analyses of the testis and ovaries of sexually mature SY-medaka, <i>dmy</i> expression was not detected. We speculate that high temperatures resulted in the loss of <i>dmy</i> in SY-medaka during evolution, or the lineage has another sex-determining gene. This study provides a valuable dataset for elucidating the mechanism underlying sex determination in <i>Oryzias</i> genus and advances research on functional genomics or reproduction biology in <i>O. curvinotus</i>.
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