Plant breeders’ knowledge of precise traits preferred by variety users would accelerate varietal turnover and widen adoption of newly developed maize varieties in Ghana. The objective of this research was to provide empirical evidence of trait preferences of farmers and other actors in the maize value chain in northern Ghana, based on which research strategies for maize improvement could be formulated. Participatory rural appraisal was conducted in 2016 to determine key traits preferred by maize value chain actors across the three regions in northern Ghana. A total of 279 maize value chain actors were interviewed. Different scoring and ranking techniques were used to assess the maize traits preferred by the different actors. Participatory variety selection trials were also conducted in the Tolon, West Gonja, Binduri, and Sissala East districts in northern Ghana from 2014 to 2016. The mother-baby trial approach was used to evaluate eight hybrids with 3000 farmers. Data on yield and agronomic performance of the hybrids and farmer’s selection criteria were collected. Data analyses were performed using GenStat Edition 16 and SPSS Edition 20 statistical packages. The participatory rural appraisal method identified farmers, input dealers, traders, and processors as the primary maize value chain actors in the study areas. Trait preferences of the different actors overlapped and revolved around grain quality including nutritional value, and stress tolerance and grain yield. Results of the participatory variety selection study revealed that across districts, farmers preferred high-yielding varieties with multiple cobs per plant, white grain endosperm color, and bigger and fully filled cobs. For the first time, our holistic assessment of the trait preferences of key actors of the maize value chain in northern Ghana revealed a comprehensive list of traits, which could be used by breeders to develop varieties that may be preferred by all value chain actors in northern Ghana.