The ability to make informed decisions on the suitability of alternative marker loci is central for population and epidemiological investigations. This issue was addressed using Staphylococcus aureus as a model population by generating nucleotide sequence data from 33 gene fragments in a representative sample of 30 strains. Supplementing the data with pre-existing multilocus sequence typing data, an intra-species tree based on similar to 17.8 kb of sequence was reconstructed and the goodness of fit of each individual gene tree was computed. No strong association was noted between gene function per se and phylogenetic reliability, but it is suggested that candidate loci should possess at least the average degree of nucleotide diversity for all genes in the genome. In the case of S. aureus this threshold is > 1% mean pairwise diversity.