Channel catfish (Ictalurus punctatus) has been recognized as a dominant freshwater aquaculture species in the United States. It is also a suitable model for studying the mechanisms of sex determination and differentiation because of its sexual plasticity and exhibition of both genetic and environmental sex determination. The testicular differentiation in male channel catfish normally starts between 90 and 102 days postfertilization (dpf), while the ovarian differentiation starts early from 19 dpf. As such, efforts to better understand the postponed testicular development at the molecular level are needed. Toward that end, we conducted transcriptomic comparison of gene expression of male and female gonads at 90, 100, and 110 dpf using high-throughput RNA-Seq. Transcriptomic profiles of male gonads on 90 and 100 dpf exhibited high similarities except for a small number of significantly up-regulated genes that were involved in development of germ cell-supporting somatic cells, while drastic changes were observed during 100–110 dpf, with a group of highly up-regulated genes that were involved in germ cells development, including nanog and pou5f1. Transcriptomic comparison between testes and ovaries identified male-preferential genes, such as gsdf, cxcl12, as well as other cytokines mediated the development of the gonad into a testis. Co-expression analysis revealed highly correlated genes and potential pathways underlying germ cell differentiation and spermatogonia stem cell development. The candidate genes and pathways identified in this study set the foundation for further studies on sex determination and differentiation in catfish as well as other teleosts.