The study of the tempo and mode of molecular evolution has played a key role in evolutionary biology, both as a stimulant for theoretical enrichment and as the foundation of useful analytical tools. When protein and DNA sequences were first produced, the surprising constancy of rates of change brought molecular evolution into conflict with mainstream evolutionary biology, but also stimulated the formation of new theoretical understanding of the processes of genetic change, including the recognition of the role of neutral mutations and genetic drift in genomic evolution. As more data were collected, it became clear that there were systematic differences in the substitution rate between species, which prompted further elaboration of ideas such as the generation time effect and the nearly neutral theory. Comparing substitution rates between species continues to provide a window on fundamental evolutionary processes. However, investigating patterns of substitution rates requires attention to potential complicating factors such as the phylogenetic non-independence of rates estimates and the time-dependence of measurement error. This chapter compares different analytical approaches to study the tempo and mode of molecular evolution, and considers the way a richer biological understanding of the causes of variation in substitution rate might inform our attempts to use molecular data to uncover evolutionary history.