Abstract Organisms within species have numerous genetic and phenotypic variations. Growing evidences show intraspecies variation of mutant phenotypes may be more complicated than expected. Current studies on intraspecies variations of mutant phenotypes are limited to just a few strains. This study investigated the intraspecies variation of fitness effects of 5,630 gene mutants in ten Saccharomyces cerevisiae strains using CRISPR–Cas9 screening. We found that the variability of fitness effects induced by gene disruptions is very large across different strains. Over 75% of genes affected cell fitness in a strain-specific manner to varying degrees. The strain specificity of the fitness effect of a gene is related to its evolutionary and functional properties. Subsequent analysis revealed that younger genes, especially those newly acquired in S. cerevisiae species, are more likely to be strongly strain-specific. Intriguingly, there seems to exist a ceiling of fitness effect size for strong strain-specific genes, and among them, the newly acquired genes are still evolving and have yet to reach this ceiling. Additionally, for a large proportion of protein complexes, the strain specificity profile is inconsistent among genes encoding the same complex. Taken together, these results offer a genome-wide map of intraspecies variation for fitness effect as a mutant phenotype and provide an updated insight on intraspecies phenotypic evolution.