We have used chromosome engineering to replace native centromeric DNA with different test sequences at native centromeres in two different strains of the fission yeast Schizosaccharomyces pombe and have discovered that A + T rich DNA, whether synthetic or of bacterial origin, will function as a centromere in this species. Using genome size as a surrogate for the inverse of effective population size (Ne) we also show that the relative A + T content of centromeric DNA scales with Ne across 43 animal, fungal and yeast (Opisthokonta) species. This suggests that in most of these species the A + T content of the centromeric DNA is determined by a balance between selection and mutation. Combining the experimental results and the evolutionary analyses allows us to conclude that A + T rich DNA of almost any sequence will function as a centromere in most Opisthokonta species. The fact that many G/C to A/T substitutions are unlikely to be selected against may contribute to the rapid evolution of centromeric DNA. We also show that a neo-centromere sequence is not simply a weak version of native centromeric DNA and suggest that neo-centromeres require factors either for their propagation or establishment in addition to those required by native centromeres.