Gene duplication is the primary source of new genes, but the mechanisms underlying the functional divergence and retention of duplicate genes are not well understood. Because eukaryotic proteins are localized to subcellular structures and localization can be altered by a single amino acid replacement, it was recently proposed that protein subcellular relocalization (PSR) plays an important role in the functional divergence and retention of duplicate genes. Although numerous examples of distinct subcellular localizations of paralogous proteins have been reported, it is unknown whether PSR occurs more frequently after gene duplication than without duplication. By analyzing experimentally determined and computationally predicted genome-wide protein subcellular localization data of the budding yeast Saccharomyces cerevisiae and two other fungi (Schizosaccharomyces pombe and Kluyveromyces waltii), we show that even singleton genes have an appreciable rate of relocalization in evolution and that duplicate genes do not relocalize more frequently than singletons. These results suggest that subcellular relocalization is unlikely to have been a major mechanism for duplicate gene retention and functional divergence at the genomic scale.