Vitellogenin gene (Vg) expression was investigated in the giant freshwater prawn, Macrobrachium rosenbergii at different ovarian stages and after exposure to 4-nonylphenol (4-NP), a widespread xeno-oestrogen present in aquatic environments. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed a high level of Vg expression in the hepatopancreas at all ovarian stages, but at a much lower level in the ovary and real-time PCR showed differential Vg expression in both the hepatopancreas and ovary. Vg expression in the hepatopancreas increased significantly at the mature ovarian stage. The expression profile in the ovary was the inverse of that in the hepatopancreas. Furthermore, the hepatopancreas was more responsive to the oestrogenic activity of 4-NP. Exposure to 50–250 μg L−1 4-NP for 96 h increased Vg expression in the hepatopancreas in a dose-dependent manner, whereas Vg expression was less in the ovary. In conclusion, Vg expression was tissue- and stage-dependent. These factors must be considered when Vg expression is used as an indicator of reproductive activity in an organism or for assessing xeno-oestrogens in the environments.