Passive integrated transponder (PIT) tagging has proven to be an effective mark-recapture technique for many temperate freshwater and marine fish species, but its adaptability to tropical freshwater species remains largely unknown. Nevertheless, many tropical river systems, such as the Mekong in South East Asia, are currently being developed at an unprecedented rate for their relatively abundant water resources. Consequently, there is an urgent need for efficient mark-recapture technologies to understand and assess the impacts of human developments on the movement ecology of tropical freshwater fish species. This paper discusses the development of an optimal protocol for PIT tagging tropical freshwater fishes, using two Mekong River species – Striped catfish (Pangasianodon hypophthalmus) and Goldfin tinfoil barb (Hypsibarbus malcolmi) – as model species. • The PIT tagging protocol is flexible in that it allows the transponders to be placed in a variety of body locations.• The protocol has high tag retention rates (>90%) and is non-invasive, since it does not affect fish growth or mortality rates.• The application of PIT tags can be used to evaluate the success of fishways and other remedial works for supporting crucial life-cycle processes potentially requiring fish passage, such as spawning.