Isolation and characterization of genes involving gonadal development are an initial step towards understanding reproductive maturation and sex determination of the giant tiger shrimp (Penaeus monodon). In the present study, 896 clones from the testis cDNA library were sequenced. A total of 606 ESTs (67.6%) significantly matched sequences in the GenBank (E-value <1e-04) whereas 290 ESTs (32.4%) were newly unidentified transcripts. The full length cDNA of genes functionally involved in testicular development including cyclophilin A (PMCYA), small ubiquitin-like modifier 1 (PMSUMO-1), ubiquitin conjugating enzyme E2, dynactin subunit 5, cell division cycle 2 (cdc2) and mitotic checkpoint BUB3 were discovered. In addition, Tra-2, a gene involving sex determination cascades, was successfully characterized by RACE-PCR and first reported in crustaceans. Expression analysis indicated that a homologue of low molecular weight neurofilament protein XNF-L (termed P. monodon testis-specific transcript 1, PMTST1; N=8 for each sex) was only expressed in testes but not ovaries. PMCYA, thyroid hormone receptor-associated protein complex 240 kDa component (Trap240), multiple inositol polyphosphate phosphatase 2 (MIPP2) and heat shock-related 70 kDa protein 2 (HSP70-2), but not PMSUMO-1, PMTra-2 and prohibitin2 were differentially expressed between ovaries and testes of P. monodon. Expression of PMTST1 was up-regulated but that of the remaining genes in testes of P. monodon broodstock was down-regulated after shrimp were molted (P<0.05). Significant reduction of PMSUMO-1 and increment of prohibitin2 transcripts in domesticated broodstock (P<0.05) suggested that these reproductively related genes may be used as biomarkers to evaluate reduced degrees of the reproductive maturation in domesticated P. monodon.