The construction of dams and other river infrastructures is threatening migratory fish stocks in many parts of the world by greatly reducing river-floodplain connectivity. Fishways are technical tools that can restore river connectivity for migratory fishes, but their effectiveness is often limited by a lack of biological information on their target species, especially in developing countries of South East Asia. We sought to inform the design of a vertical-slot fishway for Myanmar migratory fishes, by using a flume-based vertical-slot fishway to determine if (1) their passage ability is influenced by hydraulic conditions (such as depth and head differential or ‘headloss’ between cells); and (2) fish size influences passage ability for each species. This was achieved by comparing passage success under headloss options of 50-mm and 100-mm, and by assessing the relationship between size and ability to pass the flume – in four representative migratory species: Pangasius pangasius (pangas catfish), Notopterus notopterus (featherback fish), Puntius chola (swamp barbs) and Esomus danrica (flying barbs). Our results indicated that flume passage ability was greater under the 50-mm headloss for Pangasius pangasius, Notopterus notopterus and Puntius chola; and that flume passage ability increased in larger individuals for Pangasius pangasius and Notopterus notopterus. In comparison, Esomus danrica could not ascend under either the 50-mm or 100-mm headloss options. The inter-specific variability in these results empirically demonstrates the importance of obtaining baseline biological information on the target species for planned fishways in order to optimise their effectiveness.