There are increasing concerns that climatic and land use changes will enhance soil respiration rates and soil organic carbon loss, compromising agricultural productivity and further elevating atmospheric CO2. Current understanding of dryland respiration is, however, insufficient to enable prediction of the consequences of these changes for dryland soils and CO2 fluxes. The objectives of this paper are to present in-situ respiration data from five remote sites along a climatic gradient in the Kalahari of Botswana and to determine the effects of temperature, moisture and biological crust cover on soil CO2 fluxes. Moisture was the primary limiting factor to efflux which increased with amount of simulated rainfall. On dry soils, mean CO2 efflux was between 1.5 and 5.9 mg C m− 2 h− 1. After 2 mm and 50 mm simulated wetting, mean rates increased to 4.0 to 21.8 and 8.6 to 41.5 mg C m− 2 h− 1 respectively. Once wet, soil CO2 efflux increases with temperature, and sites at the hotter northern end of the transect lost more CO2 than cooler southerly sites. Net respiration rates are, however, muted by autotrophic organisms in biological soil crusts which photosynthesise and take up CO2. The temperature sensitivity of soil CO2 efflux increased with moisture. Dry, 2 mm and 50 mm treated soils had a Q10 of 1.1, 1.5 and 1.95 respectively. Our findings indicate that higher temperatures and a loss of biological crust cover will lead to greater soil C loss through respiration.