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Although being an important conservation tool in Africa, trophy hunting is known to influence risk perception in wildlife species, thus affecting the behaviour and fitness of most targeted species. We studied the effects of trophy hunting on the flight behaviour of impala (Aepyceros melampus), greater kudu (Tragelaphus strepsiceros) and sable (Hippotragus niger) in two closed ecosystems, Cawston Ranch (hunting area) and Stanley and Livingstone Private Game Reserve (tourist area), western Zimbabwe. Using standardized field procedures, we assessed the flight behavioural responses of the three species in two seasons: non-hunting (December-March) and hunting (April-November) between March 2013 and November 2014. We tested the effect of habitat, group size, sex, season, start distance and alert distance on flight initiation distance using linear mixed models. Habitat, group size sex and alert distance did not have any effect on flight initiation distance for the three species. The three species were more alert and displayed longer flight initiation distances in the hunting area compared with the tourist area. Flight initiation distances for the three species were higher during the hunting season for the hunting area and low during the non-hunting season. Flight distances of the three species did not differ between the hunting area and the tourist area. We concluded that trophy hunting increased perceived risk of wild ungulates in closed hunting areas, whereas ungulates in non-hunting areas are less responsive and somehow habituated to human presence. Management plans should include minimum approach distances by tourists as well as establishing seasonal restrictions on special zones to promote species viability. Research aimed at integrating behavioural responses with physiological aspects of target species should be promoted to ensure that managers are able to deal with the behavioural trade-offs of trophy hunting at local and regional scale.