Sterility is a serious problem that can affect all bionts. In teleosts, double haploids (DHs) induced by mitogynogenesis are often sterile. This sterility severely restricts the further application of DHs for production of clones, genetic analysis, and breeding. However, sterile DH individuals are good source materials for investigation of the molecular mechanisms of gonad development, especially for studies into the role of genes that are indispensable for fish reproduction. Here, we used the Illumina sequencing platform to analyze the transcriptome of sterile female DH Japanese flounder in order to identify major genes that cause sterility and to provide a molecular basis for an intensive study of gonadal development in teleosts. Through sequencing, assembly, and annotation, we obtained 52,474 contigs and found that 60.7% of these shared homologies with existing sequences. A total of 1225 differentially expressed unigenes were found, including 492 upregulated and 733 downregulated genes. Gene Ontology and KEGG analyses showed that genes showing significant upregulation, such as CYP11A1, CYP11B2, CYP17, CYP21, HSD3β, bcl2l1, and PRLR, principally correlated with sterol metabolic process, steroid biosynthetic process, and the Jak-stat signaling pathway. The significantly downregulated genes were primarily associated with immune response, antigen processing and presentation, cytokine-cytokine receptor interaction, and protein digestion and absorption. Using a co-expression network analysis, we conducted a comprehensive comparison of gene expression in the gonads of fertile and sterile female DH Japanese flounder. Identification of genes showing significantly different expression will provide further insights into DH reproductive dysfunction and oocyte maturation processes in teleosts.