This book constitutes the proceedings of the 21st International Conference on Comparative Genomics, RECOMB-CG 2024, which was held in Boston, MA, USA, during April 27-28, 2024. The 13 full papers presented in this book were carefully reviewed and selected from 21 submissions. The papers are divided into the following topical sections: phylogenetic networks; homology and phylogenetic reconstruction; tools for evolution reconstruction; genome rearrangements; and genome evolution.