As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.