AbstractThis study investigated the relationships between land use, habitat quality, physicochemical water quality and fish assemblage in the Sironko River catchment, a tropical mountain stream flowing into the Lake Kyoga asin in Eastern Uganda. The study was carried out at different land use sampling sites (natural forest, pastureland, plantation forest, mixed agriculture, banana plantation, urban and sugarcane plantation) for 6 months from November 2019 to April 2020. The habitat quality score (HQS) ranged between 49% in the urban area and 75% in the natural forest. There was a general increase in the temperature, conductivity, total dissolved solids, turbidity and nitrate concentration in the Sironko River as it flowed from upstream to downstream from the natural forest to the sugarcane plantation. Conversely, there was a reduced dissolved oxygen concentration and water transparency as the river traversed the different land uses downstream. A total of 765 fish individuals belonging to 9 fish species (Amphilius jacksonii, Amphilius uranoscopus, Barbus altianalis, Labeo victorianus, Clarias leocephalus, Clarias carsonii, Cyprinus carpio, Barbus palludinosus and Barbus jacksonii) were captured using an electrofishing gear. Fish identification was based on external morphological characteristics. Amphilius uranoscopus and Barbus altianalis were only caught in the natural forest and pastureland at a high altitude characterized by low temperature and high dissolved oxygen concentration. The fish species belonged to 3 trophic feeding groups dominated by the insectivorous fish. Pearson correlation coefficients showed a weak negative relationship (r = −0.249) between fish community and habitat quality, while fish assemblage and physicochemical water quality unveiled a weak positive relationship (r = 0.176). The coefficient of determination (R2 = 0.695) indicated 69.5% of the fish community variability was accounted for by the physicochemical water quality variables in Sironko River System. Accordingly, it was concluded that physicochemical water quality attributes are the most important factors determining the distribution of fish species with land use in the Sironko River catchment, a tropical mountain stream. The Sironko River catchment is hydraulically and biologically connected to Lake Kyoga through a series of satellite lakes and swamps. The river is spawning area for Clarias leocephalus, Clarias carsonii and Amphilius lujani fish species of the lake.