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The nucleosome is the fundamental structural units of eukaryotic chromatin. During mitosis, duplicated nucleosome fibers are organized into a pair of rod-shaped structures (chromatids) within a mitotic chromosome. However, it remains unclear whether nucleosome assembly is indeed an essential prerequisite for mitotic chromosome assembly. Here, we combined mouse sperm nuclei and Xenopus cell-free egg extracts depleted of the histone chaperone Asf1 and found that chromatid-like structures could be assembled even in the near-absence of nucleosomes. The resultant “nucleosome-depleted” chromatids contained discrete central axes positive for condensins, although they were more fragile than normal nucleosome-containing chromatids. Combinatorial depletion experiments underscored the central importance of condensins in mitotic chromosome assembly, which sheds light on their functional crosstalk with nucleosomes in this process.