As part of the South African National Survey of Arachnida (SANSA), spiders were collected from all the field layers in the Polokwane Nature Reserve (Limpopo Province, South Africa) over a period of a year (2005–2006) using four collecting methods. Six habitat types were sampled: Acacia tortillas open savanna; A. rehmanniana woodland, false grassland, riverine and sweet thorn thicket, granite outcrop; and Aloe marlothii thicket. A total of 13 821 spiders were collected (using sweep netting, tree beating, active searching and pitfall trapping) represented by 39 families, 156 determined genera and 275 species. The most diverse families are the Thomisidae (42 spp.), Araneidae (39 spp.) and Salticidae (29 spp.). A total of 84 spp. (30.5%) were web builders and 191 spp. (69.5%) wanderers. In the Polokwane Nature Reserve, 13.75% of South African species are presently protected.