This book constitutes the refereed proceedings of the 20th Annual RECOMB Satellite Workshop on Comparative Genomics, RECOMB-CG 2023 which took place in Istanbul, Turkey, in April 2023.  The 15 full papers included in this book were carefully reviewed and selected from 25 submissions. The papers present cutting edge research in comparative genomics, with an emphasis on computational approaches and novel experimental results. Chapters "Inferring Clusters of Orthologous and Paralogous Transcripts" and "Gene Order Phylogeny via Ancestral Genome Reconstruction under Dollo" are published Open Access under Creative Commons Attribution license (CC BY 4.0).