We report five novel anonymous nuclear loci for the African viper genus Bitis, developed from a genomic library of a single individual puff adder (Bitis arietans). All loci amplify consistently and are variable among seven B. arietans sampled from disparate portions of the geographic range. Trials with nine congeneric species showed one locus amplifies across all species tested, three amplify in a subset of species and one only amplifies in B. arietans. Genetic divergences for these loci range from comparable, to more than double that of a commonly used nuclear protein-coding marker in Bitis. These loci will provide a valuable resource for future investigations of these snakes.