ABSTRACT: : The giant freshwater prawn (Macrobrachium rosenbergii) exhibits sex dimorphism between the male and female individuals. To date, the molecular mechanism governing gonadal development was unclear, and limited data were available on the gonad transcriptome of M. rosenbergii. Here, we conducted comprehensive gonadal transcriptomic analysis of female (ZW), super female (WW), and male (ZZ) M. rosenbergii for gene discovery. A total of 70.33 gigabases (Gb) of sequences were generated. There were 115,338 unigenes assembled with a mean size of 1,196 base pair (bp) and N50 of 2,195 bp. Alignment against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide/protein sequence database (NR and NT), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, SwissProt database, Protein family (Pfam), Gene ontology (GO), and the eukaryotic orthologous group (KOG) database, 36,282 unigenes were annotated at least in one database. Comparative transcriptome analysis observed that 10,641, 16,903, and 3,393 genes were significantly differentially expressed in ZW vs. ZZ, WW vs. ZZ, and WW vs. ZW samples, respectively. Enrichment analysis of differentially expressed genes (DEGs) resulted in 268, 153, and 42 significantly enriched GO terms, respectively, and a total of 56 significantly enriched KEGG pathways. Additionally, 23 putative sex-related genes, including Gtsf1, IR, HSP21, MRPINK, Mrr, and other potentially promising candidate genes were identified. Moreover, 56,241 simple sequence repeats (SSRs) were identified. Our findings provide a valuable archive for further functional analyses of sex-related genes and future discoveries of underlying molecular mechanisms of gonadal development and sex determination.