Larimichthys crocea is an economically important marine fish in China. To date, the molecular mechanisms underlying testicular development and spermatogenesis in L. crocea have not been thoroughly elucidated. In this study, we conducted a comparative transcriptome analysis between testes (TES) and pooled multiple tissues (PMT) (liver, spleen, heart, and kidney) from six male individuals. More than 54 million clean reads were yielded from TES and PMT libraries. After mapping to the draft genome of L. crocea, we acquired 25,787 genes from the transcriptome dataset. Expression analyses identified a total of 3853 differentially expressed genes (DEGs), including 2194 testes-biased genes (highly expressed in the TES) and 1659 somatic-biased genes (highly expressed in the PMT). The dataset was further annotated by blasting with multi-databases. Functional genes and enrichment pathways involved in spermatogenesis and testicular development were analyzed, such as the neuroactive ligand-receptor interaction pathway, gonadotropin-releasing hormone (GnRH) and mitogen-activated protein kinase (MAPK) signaling pathways, cell cycle pathway, and dynein, kinesin, myosin, actin, heat shock protein (hsp), synaptonemal complex protein 2 (sycp2), doublesex-and mab-3-related transcription factor 1 (dmrt1), spermatogenesis-associated genes (spata), DEAD-Box Helicases (ddx), tudor domain-containing protein (tdrd), and piwi genes. The candidate genes identified by this study lay the foundation for further studies into the molecular mechanisms underlying testicular development and spermatogenesis in L. crocea.Genes 2019, 10, 958 2 of 22 remains for somatic growth; these changes eventually reduce the culture benefit of L. crocea [3] and hinder the conservation of its germplasm resources. Therefore, the molecular mechanisms underlying reproduction regulation must be investigated, and the genes involved in gametogenesis and gonadal development must be identified, to provide a theoretical reference for further studies on the above problems in L. crocea.Directly correlated with sexual maturation and reproduction, in various animal species, including L. crocea, the testis is an essential basic component of the animal reproductive system and responsible for the production of male gametes via spermatogenesis [4]. During the process of spermatogenesis, diploid spermatogonia slowly evolve into many highly specialized spermatozoa through mitosis, meiosis, and spermiogenesis. Stringent spatial and temporal expression of genes during both transcriptional and translational processes are fundamental to ensure the precise processes of spermatogenesis [5]. Previous studies have focused on the reproductive and developmental biology of L. crocea based on anatomical/histological aspects [6,7], whereas few studies have focused on genes related to spermatogenesis and testicular development. To the best of our knowledge, only a small number of reproduction-related genes have been identified in L. crocea [8][9][10][11]. To further comprehensively explore the mol...