One of the aims of South African National Survey of Arachnida (SANSA) is to survey protected areas to obtain species-specific information and compile inventories to determine species distribution patterns and evaluate their conservation status for Red Data assessments. The aim of this study, the first in a series of surveys of the Diamond Route Reserves, was to compile the first checklist of the spider species in the Northern Cape at the Tswalu Kalahari Reserve. Spiders were collected during three survey periods (2005−2013) using different collecting methods to sample both the ground and field layers. In total, 32 families represented by 108 genera and 136 species have been collected so far. The most species-rich families are the Salticidae (20 spp.) and Thomisidae (18 spp.), followed by the Gnaphosidae and Araneidae (11 spp. each), while nine families are represented by singletons. The free-living wandering spiders represent 97 spp., while 39 spp. are web-builders. Information on spider guilds, endemicity value and conservation status are provided. The Tswalu Kalahari Reserve protects approximately 6.1% of the total South African spider fauna, while 24.3% of the species found in the reserve are South African endemics, of which 5.9% are Northern Cape endemics. Approximately 6.0% of the species sampled are possibly new to science or represent new records for South Africa.