Hainan medaka (Oryzias curvinotus) 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 O. curvinotus in Sanya River (SY-medaka), which lacks dmy (a male sex-determining gene in O. latipes and O. curvinotus). 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 O. curvinotus. A mitochondrial cytochrome oxidase subunit I (CoI) sequence alignment showed that the sequence identities between SY-medaka and other geographic populations of O. curvinotus are as high as 95%. A phylogenetic analysis of the mitochondrial genome also indicated that SY-medaka belongs to O. curvinotus. Molecular marker-based genetic sex assays and whole genome re-sequencing showed that SY-medaka does not contain dmy. Further, in RNA-Seq analyses of the testis and ovaries of sexually mature SY-medaka, dmy expression was not detected. We speculate that high temperatures resulted in the loss of dmy 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 Oryzias genus and advances research on functional genomics or reproduction biology in O. curvinotus.