Hemibarbus maculatus is a globally important species for its value in fisheries and aquaculture. There is relatively little information available about this species' sexual development genes. Therefore, in this study, we used the technology of high‐throughput RNA sequencing (RNA‐seq), to investigate if the ovary and testis' differently expressed genes (DEGs) during sex differentiation in H. maculatus. Our results revealed a total of 43,468,248 and 39,241,362 qualified reads were generated in the female and male libraries, and 32,127 unigenes were assembled via the annotation analysis. Moreover, 17,808 unigenes were found to be DEGs between males and females, with 12,726 up‐ and 5082 downregulated genes in the ovaries and testes, respectively. The transcriptome analysis revealed there are 23 DEGs between ovary and testes of H. maculatus, which may be involved in sex determination. The expression patterns of these genes were validated by quantitative real‐time PCR (qPCR). The results of qPCR were generally consistent with the RNA‐seq results. This work was the first to annotate genes linked with gonadal development and reproduction in H. maculatus using gonadal transcriptome analysis, which provides useful molecular data for a better understanding of sexual development, as well as functional genomics and population genetics studies of H. maculatus.