In this study, the relative role of spatio-temporal factors and associated environmental variables (water transparency and temperature) were quantified in relation to gillnet samples of fishes in a large and shallow lake (Lake Balaton, Hungary). Most of the variance (56·1%) in the relative abundance data (%) was related to the vertical segregation of fishes. This gradient substantially affected the catch per unit effort (CPUE) by number of the dominant species, the surface-oriented bleak Alburnus alburnus and the benthic common bream Abramis brama. It also influenced total CPUE, mean fish mass and species richness and diversity. At the lake level, horizontal habitat heterogeneity (i.e. littoral v. offshore) accounted for only 8·3% of the total variance in relative abundance data, but was important in structuring the CPUE of the ruffe Gymnocephalus cernua and the pikeperch Sander lucioperca. The longitudinal environmental gradient (i.e. lake basin), year and season of sampling, water transparency and temperature had significant effects on relative abundance only at the habitat level, but were also important components of variability of CPUE in some species at the lake level. As sampling schemes need to consider the main gradients in fish assemblage distributions, the use of surface and pelagic gillnets should be more intensively incorporated in the study and monitoring of fish assemblages in shallow lakes and lake habitats.