Gender is well recognised as a critical consideration for HIV/AIDS organisations. Since the 1990s, HIV/AIDS policy-makers, donors, non-governmental organisations and transnational corporations have adopted gender mainstreaming as the process for integrating gender into development programmes and institutions. There is an increasing body of literature on the successes and challenges of practicing gender mainstreaming within organisational environments, however, little has been said about this practice within HIV/AIDS-specific organisational environments. As a contribution to this gap, this reflective paper aims to generate debate about some of the considerations for gender mainstreaming practice in HIV/AIDS organisations. It draws on the author's experience conducting a gender mainstreaming review with a southern African HIV/AIDS capacity-strengthening organisation, as well as a review of the development literature on gender mainstreaming. The paper looks at three key issues facing gender mainstreaming: (1) donor requirements on disaggregating data by sex; (2) connecting gender mainstreaming with the priorities of community HIV/AIDS organisations; and (3) the role of resistance to gender mainstreaming as neo-colonial. Preliminary understandings of these issues suggest that current approaches to gender mainstreaming may not be flexible enough to consider the multiple ways gender and HIV/AIDS interact in different sociocultural contexts. There is an urgent need for further debate and in-depth research into these issues, given the challenge they pose for HIV/AIDS organisations and donors that have chosen to make gender mainstreaming a criterion for HIV/AIDS funding.