AbstractDiploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications—doublings of the entire complement of chromosomes—are linked to genetic instability and frequently found in human cancers^1–3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis^4–8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis⁹ and DNA double-strand breaks¹⁰. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.