In order to clone and analyze the differentially expressed genes of gonads of domesticated broodstock Penaeus monodon Fabricius，total RNA was extracted from the ovary and testis，and differential display technique was performed to screen the differentially expressed genes by using a set of 3 anchored primers for cDNA synthesis and 8 arbitrary primers in the PCR. The differential display DNA bands were separated by denaturing ureapolyacrylamide gel electrophoresis (PAGE) and displayed by silver staining. Finally 173 cDNA fragments were isolated，and 44 cDNA fragments were excised. After cloning and sequencing，11 differences in expression of the screened cDNA fragments were confirmed by real-time RT-PCR，and 10 cDNA fragments proved to be real difference. Results show that these 10 fragments were expressed differently between ovary and testis，7 fragments were expressed significantly higher level in ovary，two fragments were expressed significantly higher level in testis，one fragment was particularly expressed in testis. The nucleotide sequence analysis revealed that two fragments were not homologous to of the known genes or ESTs，may be novel genes. TC1 was only expressed in testis，and there was no any difference between female and male DNA level. TG6-3 revealed a preferential expression level in testes than ovaries，and it was only expressed in male brain but no expression in female brain. This research provided a foundation for further studies of the molecular mechanism of sexual differentiation and gonadal development of Penaeus monodon.