Anatoxins are carcinogenic mycotoxins produced by Aspergillus spp. In groundnut kernels. Forty-six groundnut samples were collected from subsistence farmers In three provinces of South Africa, namely KwaZulu-Natal (KZN), Mpumalanga (MP) and Limpopo (LP), in 2006 and 2007. Anatoxin levels of groundnut kernels were quantified using an ELISA technique. The occurrence of A. parasiticus colonies was predominant and it was isolated at twice the frequency of A. flavus. Anatoxins were present in groundnut produced in the northern parts of KZN, where aflatoxin levels of up to 131 parts per billion (ppb) were found. In Mpumalanga and Limpopo, the highest aflatoxin levels were 160 ppb and 2 ppb, respectively. In the Makhanisi and Mbazwane localities situated in KwaZulu-Natal, and in Boshoffontein in Mpumalanga, aflatoxin levels were higher than the maximum permitted level set by the Food and Drug Administration in the USA (20 ppb), the European Union (6 ppb) and the Department of Health in South Africa (10 ppb) for groundnut that is Intended for direct human consumption. This study indicates the need for mycotoxin awareness campaigns and control programs to be implemented in rural areas of South Africa.Articl