When homologous microsatellites are compared between species, significant differences in mean length are often noted. A dominant cause of these length differences is ascertainment bias due to selection for maximum repeat number and repeat purity when the markers are being developed. However, even after ascertainment bias has been allowed for through reciprocal comparisons, significant length differences remain, suggesting that the average microsatellite mutation rate differs between species. Two classes of mechanism have been proposed: rapid evolution of enzymes involved in the generation and repair of slippage products (enzyme evolution model) and heterozygote instability, whereby interchromosomal events at heterozygous sites offer extra opportunities for mutations to occur (heterozygote instability model). To examine which of these hypotheses is most likely, we compared ascertainment bias and species length differences between humans and chimpanzees in autosomal and Y chromosomal microsatellites. We find that levels of ascertainment bias are indistinguishable, but that interspecies length differences are significantly greater for autosomal loci compared with haploid Y chromosomal loci. Such a pattern is consistent with predictions from the heterozygote instability model and is not expected under models of microsatellite evolution that do not include interchromosomal events such as the enzyme evolution model.