Yeast cells are extensively used as a key model organism owing to their highly conserved genome, metabolic pathways, and cell biology processes. To assist in genetic engineering and analysis, laboratory yeast strains typically harbor auxotrophic selection markers. When uncompensated, auxotrophic markers cause significant phenotypic bias compared to prototrophic strains and have different combinatorial influences on the metabolic network. Here, we used BY4741, a laboratory strain commonly used as a “wild type” strain in yeast studies, to generate a set of revertant strains, containing all possible combinations of four common auxotrophic markers (leu2∆, ura3∆, his3∆1, met15∆). We examined the effect of the auxotrophic combinations on complex phenotypes such as resistance to rapamycin, acetic acid, and ethanol. Among the markers, we found that leucine auxotrophy most significantly affected the phenotype. We analyzed the phenotypic bias caused by auxotrophy at the genomic level using a prototrophic version of a genome‐wide deletion library and a decreased mRNA perturbation (DAmP) library. Prototrophy was found to suppress rapamycin sensitivity in many mutants previously annotated for the phenotype, raising a possible need for reevaluation of the findings in a native metabolic context. These results reveal a significant phenotypic bias caused by common auxotrophic markers and support the use of prototrophic wild‐type strains in yeast research.