Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show that rRNA species associate with condensed chromosomes during mitosis. In particular, pre-rRNAs such as 45S, 32S, and 30S are highly enriched on mitotic chromosomes. Immediately following nucleolus disassembly in mitotic prophase, rRNAs are released and associate with and coat each condensed chromosome at prometaphase. Using unbiased mass spectrometry analysis, we further demonstrate that chromosome-bound rRNAs are associated with Ki-67. Moreover, the FHA domain and the repeat region of Ki-67 recognize and anchor rRNAs to chromosomes. Finally, suppression of chromosome-bound rRNAs by RNA polymerase I inhibition or by using rRNA-binding-deficient Ki-67 mutants impair mitotic chromosome dispersion during prometaphase. Our study thus reveals an important role of rRNAs in preventing chromosome clustering during mitosis.