The Ki-67 protein is a widely used marker of mammalian cell proliferation that plays critical roles in heterochromatin organisation and mitotic perichromosomal layer formation. Thus, its apparent restriction to vertebrates is puzzling. However, sequences with limited homology to subdomains of Ki-67 are present in genomes from some non-vertebrate species. Here, using structural modelling, biophysical and functional analysis, we identify Ki-67-like proteins across diverse non-vertebrate eukaryotes