This book constitutes the refereed proceedings of the 27th International Symposium on String Processing and Information Retrieval, SPIRE 2020, held in Orlando, FL, USA, in October 2020.The 17 full papers and 4 short papers presented in this volume were carefully reviewed and selected from 32 submissions. They cover topics such as: data structures; algorithms; information retrieval; compression; combinatorics on words; and computational biology.