BACKGROUND: Recent research has indicated a positive association between rates of molecular evolution and diversification in a number of taxa. However debate continues concerning the universality and cause of this relationship. Here, we present the first systematic investigation of this relationship within the mammals. We use phylogenetically independent sister-pair comparisons to test for a relationship between substitution rates and clade size at a number of taxonomic levels. Total, non-synonymous and synonymous substitution rates were estimated from mitochondrial and nuclear DNA sequences. RESULTS: We found no evidence for an association between clade size and substitution rates in mammals, for either the nuclear or the mitochondrial sequences. We found significant associations between body size and substitution rates, as previously reported. CONCLUSIONS: Our results present a contrast to previous research, which has reported significant positive associations between substitution rates and diversification for birds, angiosperms and reptiles. There are three possible reasons for the differences between the observed results in mammals versus other clades. First, there may be no link between substitution rates and diversification in mammals. Second, this link may exist, but may be much weaker in mammals than in other clades. Third, the link between substitution rates and diversification may exist in mammals, but may be confounded by other variables.