This paper presents the results of a faunistic study of the macrozoobenthos of the Velika Morava River. The investigation was conducted during the summer and autumn months in 2010. A total of 84 macroinvertebrate taxa have been identified, with Insecta (Ephemeroptera) as the most diverse and Oligochaeta as the most abundant groups. A tubificid worm, Limnodrilus hoffmeisteri, was the most important species with regard to relative abundance and frequency of occurrence. Two rare and endangered species, Theodoxus transversalis and Unio crassus, were recorded, as well as 5 alien species. Locality VM4 (Markovac Bridge) is of particular interest as the northernmost locality, as well as having the most abundant population of T. transversalis found. Despite being in the lower stretch of the river, this site is particularly taxa-rich, presumably due to conspicuous microhabitat diversity. Water temperature and pH value were determined to be the most important factors of the 32 environmental variables tested. Multivariate analyses revealed separation of summer samples compared to autumn. The Mann-Whitney test showed significant differences in fauna only in the case of ecoregions, confirming their current delineation and the transitional character of this river.