Participatory action research across food value chains (FVC) can help stabilise the food security of subsistence farmers by implementing upgrading strategies (UPS). These strategies can be assessed ex-ante and ex-post for their potential social, ecological and economic sustainability impacts. UPS implementation, however, often entails gender-specific changes and challenges in a farmer's social life, economy and environment that either were not perceived and anticipated beforehand or are not followed up during UPS implementation. Before and during their implementation, therefore, UPS need to be entirely understood and assessed by both genders in terms of their potential social, ecological and economic sustainability impacts. This article conceptualises a systematic framework for integrating gender in sustainability impact assessments and presents gender-based assessment differences in three low-input UPS in Tanzanian FVC. We conducted ex-ante and ex-post impact assessments using nine food security criteria developed earlier by the authors following the Framework of Participatory Impact Assessment (FoPIA). Sustainability impact assessments—to a greater extent than expected—differed to various extents between the genders for a) different food security criteria, b) different sustainability dimensions (economic, social, and environmental), c) different points in time (T0, T1) of assessments, d) different implemented UPS, and e) different members within the groups of female and/or male stakeholders. The results demonstrate the substantial importance of integrating female-male segregated assessments and perceptions before and while implementing food-securing UPS. We anticipate that integrating these assessments and perceptions as regular components will lead to better gendered social learning for both scientists and stakeholders and a holistic understanding of complex local food systems. •A gender-focussed analytical framework for food security impact assessments is presented.•Assessments of social, ecological and economic impacts differed between the sexes.•Ex-ante and ex-post assessments differed between the sexes.•Both female and male farmers need to be integrated during UPS development.•The framework promotes gendered social learning by both scientists and stakeholders.