Abstract The fitness effects of classes of DNA mutations can be inferred from patterns of nucleotide variation. A number of studies have attributed differences in levels of polymorphism and divergence between silent and replacement mutations to the action of natural selection. Here, I investigate the statistical power to detect directional selection through contrasts of DNA variation among functional categories of mutations. A variety of statistical approaches are applied to DNA data simulated under Sawyer and Hartl's Poisson random field model. Under assumptions of free recombination and stationarity, comparisons that include both the frequency distributions of mutations segregating within populations and the numbers of mutations fixed between populations have substantial power to detect even very weak selection. Frequency distribution and divergence tests are applied to silent and replacement mutations among five alleles of each of eight Drosophila simulans genes. Putatively “preferred” silent mutations segregate at higher frequencies and are more often fixed between species than “unpreferred” silent changes, suggesting fitness differences among synonymous codons. Amino acid changes tend to be either rare polymorphisms or fixed differences, consistent with a combination of deleterious and adaptive protein evolution. In these data, a substantial fraction of both silent and replacement DNA mutations appear to affect fitness.