This study aimed to develop a set of SNP markers with high resolution and accuracy within the African buffalo. Such a set can be used, among others, to depict subtle population genetic structure for a better understanding of buffalo population dynamics. In total, 18.5 million DNA sequences of 76 bp were generated by next generation sequencing on an Illumina Genome Analyzer II from a reduced representation library using DNA from a panel of 13 African buffalo representative of the four subspecies. We identified 2534 SNPs with high confidence within the panel by aligning the short sequences to the cattle genome (Bos taurus). The average sequencing depth of the complete aligned set of reads was estimated at 5x, and at 13 x when only considering the final set of putative SNPs that passed the filtering criterion. Our set of SNPs was validated by PCR amplification and Sanger sequencing of 15 SNPs. Of these 15 SNPs, 14 amplified successfully and 13 were shown to be polymorphic (success rate: 87%). The fidelity of the identified set of SNPs and potential future applications are finally discussed.