Polymorphisms within the promoter region of the TNF-alpha gene have been associated with altered expression of TNF-alpha and susceptibility to a variety of diseases in humans. Although macaques (Macaca spp.) are frequently used as models to study human diseases, little is known about the extent of genetic variation at the TNF-alpha locus and its consequences for disease susceptibility in these species. The TNF-alpha promoter region was sequenced in a sample of 40 macaques including five M. mulatta of Chinese and Indian ancestry and 35 M. fascicularis of Malaysian, Mauritian, Indonesian, and Philippine ancestry. These groups were chosen because they exhibit differences in their susceptibilities to severe malaria upon infection with Plasmodium parasites. Sequence analysis revealed a total of 14 single nucleotide polymorphisms (SNPs), five of which are newly described, and 20 unique haplotypes. In addition, the TFSEARCH program was used to investigate the potential of these polymorphisms to influence transcription factor binding. While both species exhibited a similarly high degree of genetic variability at the TNF-alpha promoter, AMOVA analysis and FST values indicated that most of the variation is shared between species and among populations. However, two of the most common haplotypes, describing 31.7% of the observed variation, and three potentially functional polymorphisms at positions -781, -535, and -10, were exclusive to M. fascicularis. Polymorphisms in the human ortholog of the TNF-alpha promoter which are known to be associated with malaria susceptibility in humans were not shared with macaques.