Lacustrine goby (Gobiopterus lacustris) belongs to a genus of gobies that are small in size and endemic to freshwater, brackish waters or coastal environments around the Indian and Pacific oceans. G. lacustris has almost transparent skin and clearly visible internal organs, making it an ideal fish model for potential research in development, reproduction and other fields. Here, we constructed gonadal transcriptomes of G. lacustris using Illumina sequencing for the first time and identified genes that may be involved in gonadal development, gametogenesis and reproduction. Row reads were assembled into 62 573 unigenes with N50 value of 3 082 bp and a mean length of 1 869 bp. 41 480, 32 848, 37 500, 35 394, 18 318, 35 394 and 27 009 unigenes were successfully annotated in NR, NT, SwissProt, PFAM, KOG, GO and KEGG databases, respectively. Gene expression patterns in the ovary and testis were compared, and 10 954 differentially expressed genes (DEGs) were identified. Among these genes, 8 571 were up-regulated in the ovary, and 2 383 were up-regulated in the testis. qPCR analysis of 14 selected genes showed patterns consistent with the transcriptome results. Numerous DEGs involved in gonadal development and gametogenesis were identified, including foxl2, dmrt1, cyp19a1a, inha, inhb, sycp2, zglp1, tdrp, zps and esra. Using GO and KEGG enrichment analyses, pathways involved regulation of gonadal development and gametogenesis, such as "ubiquitin mediated proteolysis", "oocyte meiosis", "progesterone-mediated oocyte maturation", "p53 signaling pathway" and "PI3K-Akt signaling pathway", were also identified. In addition, 38 550 simple sequence repeats were identified from 20 517 SSR containing sequences, and 192 450 single nucleotide polymorphisms were detected. This study denotes the first gonadal transcriptomic analysis of G. lacustris and provides a valuable dataset for future functional analysis of sex-associated and molecular marker-assisted selections in G. lacustris.