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.