Spiders were collected by sifting leaf litter of three common tree species, Cussonia paniculata sinuata, Olea europaea africana and Searsia lancea, in an undisturbed grassland habitat in central South Africa. The study aimed to collect baseline data on spider assemblage structure, seasonality and the effects of litter structure on spider assemblages. In total, 1385 spiders were collected, representing 26 families and 56 species. Four species, Obatala sp. (Amaurobiidae, 22.7%), Camillina maun Platnick & Murphy (Gnaphosidae, 17.0%), Proevippa sp. (Lycosidae, 11.4%) and Zelotes frenchi Tucker (Gnaphosidae, 9.2%) dominated the fauna. Spider abundance was the highest in Olea litter (n = 607), followed by Searsia (n = 453) and Cussonia (n = 325). Searsia had the greatest number of species (41 spp.), followed by Olea (35 spp.) and Cussonia (30 spp.). Both spider abundance and species richness per sample varied significantly between the tree species. Of the ten most abundant species, all but two showed a significant preference for particular litter types. Mean spider abundance and species richness per sample were the lowest in Cussonia litter, which was the deepest litter type, but with the most interstitial space, whereas mean abundance and species richness per sample were the highest in Olea litter, which was the shallowest and moderately compact in structure.