The TP53 gene is frequently mutated in human cancer. Research has focused predominantly on six major “hotspot” codons, which account for only ∼30% of cancer-associated p53 mutations. To comprehensively characterize the consequences of the p53 mutation spectrum, we created a synthetically designed library and measured the functional impact of ∼10,000 DNA-binding domain (DBD) p53 variants in human cells in culture and in vivo. Our results highlight the differential outcome of distinct p53 mutations in human patients and elucidate the selective pressure driving p53 conservation throughout evolution. Furthermore, while loss of anti-proliferative functionality largely correlates with the occurrence of cancer-associated p53 mutations, we observe that selective gain-of-function may further favor particular mutants in vivo. Finally, when combined with additional acquired p53 mutations, seemingly neutral TP53 SNPs may modulate phenotypic outcome and, presumably, tumor progression. •Multiplexed quantification of the effect of thousands of distinct p53 mutations•Differential mutation outcomes reflect functional evolutionary constraints•Neutral SNPs may interact with acquired mutations to modulate phenotypic outcome•Hotspot p53 mutations confer a relative competitive advantage in vivo Are all p53 mutations alike? Kotler et al. compare the functional impact of thousands of variations in the p53 tumor suppressor protein in human cells in vitro and in vivo and characterize the differential outcome of distinct p53 mutations.