Because organisms respond to the environment at different scales, it is important to develop ways of determining the appropriate scales for a specific ecological process and organism. We consider whether the relative importance of different scales is associated with organism mobility, and whether this relationship is independent of landscape characteristics. We observed abundances of particular species for vascular plants, ground-dwelling beetles and breeding birds along eight 2-km transects of 40 sampling stations each, distributed over four sites along the regional gradient from shortgrass steppe in central Colorado to tallgrass prairie in central Kansas. For each transect and taxonomic group, the relative importance of factors measured at the trap scale (1 m; soil texture and hardness, vegetation height, bare ground), at the local scale (10 m; density of shrubs and cacti) and at the landscape scale (30 m; Landsat 7 TM spectral bands, slope and elevation) was assessed using hierarchical canonical variance partitioning with forward selection of explanatory variables. Plant, beetle and bird community composition was explained by environmental factors measured at all three scales. Factor influence was more consistent between transects and between plants and beetles for the more homogeneous landscapes of the shortgrass steppe than for the more heterogeneous landscapes of the tallgrass prairie. We conclude that, independent of the mobility of a taxonomic group, factors at several scales are important in explaining community composition. The importance of different scales shifts along a regional gradient, and the variability between sites is high even for nearby sites.