Tribal women play a crucial role in agro-biodiversity management and especially in subsistence food crops. Small millets are a group of cereals that are mostly underutilized and even neglected. They are primarily cultivated for food on a subsistence scale in the vulnerable agroecosystems. In many regions of the world, women cultivate such crops to meet the household food requirements. However, their contribution is not commonly recognized in in situ on-farm conservation initiatives. A study was carried out among the Malayalar tribal community in Kolli Hills, South India to understand the gender roles, decision-making, access to resources and differential knowledge among men and women who grow little amounts of millet. Gender analysis portrays how women’s work and responsibility are closely connected to the production of small millets and how the gender differentiated local knowledge plays a decisive role in managing and enhancing genetic resources. The study on the gender differential knowledge explicitly highlights the fact that knowledge acquisition differs with the need, access, and utilization of the resources. Though women play a key role, possess specialized knowledge and skill in managing millet species, their right to access and control over land resources are limited. Such a detailed understanding of the different gendered practices and associated knowledge was helpful to roll out an on-farm conservation management strategy.