The negative correlation between the rate of protein evolution and expression level of a gene has been recognized as a universal law of the evolutionary biology (Koonin 2011). In our study, we apply a population-based approach to systematically investigate the relative importance of unequal mutation rate, linkage, and selection in the origin of the expression-polymorphism anticorrelation. We analyzed the DNA sequence of protein coding genes of 24 Saccharomyces cerevisiae and 58 Schizosaccharomyces pombe strains. We found that highly expressed genes had a substantially decreased number of polymorphic sites when compared with genes transcribed less extensively. This expression-dependent reduction was especially strong in the nonsynonymous sites, although it was also present in the synonymous sites and untranslated regions, both up and down of a gene. Most importantly, no such trend was found in introns. We used these observations, as well as analyses of site frequency spectra and data from mutation accumulation experiments, to show that the purifying selection acting on nonsynonymous sites was the main, but not exclusive, factor impeding molecular evolution within the coding sequences of highly expressed genes. Linkage could not fully explain the observed pattern of polymorphism within the untranslated regions and synonymous sites, although the contribution of selection acting directly on synonymous variants was extremely small. Finally, we found that the impact of mutational bias was rather negligible.