‘Matooke’, a staple food made from Highland cooking bananas in the Great Lakes region of East Africa has been a focus for genetic improvement for resistance to pests and diseases. However, the limited information on user-preferred quality traits have led to sub-optimal adoption of new varieties and their subsequent impact on food security in the region. A gendered study to identify characteristics preferred by users who produce, process, consume and market matooke was conducted using survey data from 123 farmers, 14 focus group discussions and 40 traders in two matooke growing districts of Mbarara and Nakaseke in Uganda. The study showed that majority of the farmers sourced their planting material from their own farm, and a small percentage from government initiated programs. Limited adoption and use of improved varieties was attributed to their inferior taste compared to the land races, poor soils, lack of awareness and limited access to introduced improved varieties. There were minimal geographical and gender differences in preferences. Preferred characteristics for raw cooking banana included big bunch, big fruits, and mature bunches and green peel colour. Yellow pulp colour which is an indicator of good food is preferred. Desired characteristics for cooked matooke include soft texture, good taste, good aroma. Other traits related to marketing included compactness for easy stacking during transportation, while quality is lowered by bruising, quick ripening, and fingers that fall off easily. The increased demand for matooke was attributed to population growth and competitive prices which fluctuated with different seasons such as festive season and school term. Therefore, there is need for accurate demand-led breeding programs that incorporate user-preferred quality traits to increase adoption and impact of improved banana varieties.