Enhancers change rapidly during evolution, but the mechanism by which new enhancers originate in the genome is mostly unknown. We find that DNA replication time in the germline is tightly coupled to the emergence of new enhancers across evolution. While enhancers are most abundant at euchromatic regions, new enhancers appear nearly twice as often at late compared with early replicating regions in mammals. This is independent of transposable elements which have been linked to enhancer turnover and expansion. New enhancers are enriched in eQTLs, appear neutrally evolving and are also more likely to be tissue specific than evolutionarily conserved enhancers. Genes that bind to these enhancers, inferred via binding motifs, were also more recently evolved, and are more likely to show tissue-specific expression. Mutational biases underlying replication timing has a profound impact in shaping the classes of TFs that bind at mammalian enhancers. The association between enhancer turnover and late DNA replication time is also observed in cancer. Independent of replication time, enhancers gained or lost in cancer have accrued a higher number of mutations than unchanged enhancers implying mutations played a causal role. The similar finding across species and in cancer suggest that this is a time-invariant principle of regulatory element turnover. These results have implications for inferring the phenotypic effects of regulatory variation.