Mpox is an emerEU Horizon 2020 project VEO. Grant Number: 8EU Horizon 2020 project VEO. Grant Number: 874735 NOW Stevin prize (Koopmans) Department of Medical Microbiology and Infection Prevention of the Amsterdam UMC74735 NOW Stevin prize (Koopmans) Department of Medical Microbiology and Infection Prevention of the Amsterdam UMCging zoonotic disease which has now spread to over 113 countries as of August 2023, with over 89,500 confirmed human cases. The Netherlands had one of the highest incidence rates in Europe during the peak of the outbreak. In this study, we generated 158 near-complete mpox virus (MPXV) genomes (12.4% of nationwide cases) that were collected throughout the Netherlands from the start of the outbreak in May 2022 to August 2023 to track viral evolution and investigate outbreak dynamics. We detected 14 different viral lineages, suggesting multiple introductions followed by rapid initial spread within the country. The estimated evolutionary rate was relatively high compared to previously described in orthopoxvirus literature, with an estimated 11.58 mutations per year. Genomic rearrangement events occurred at a rate of 0.63% and featured a large deletion event. In addition, based on phylogenetics, we identified multiple potential transmission clusters which could be supported by direct source- and contact tracing data. This led to the identification of at least two main transmission locations at the beginning of the outbreak. We conclude that whole genome sequencing of MPXV is essential to enhance our understanding of outbreak dynamics and evolution of a relatively understudied and emerging zoonotic pathogen. ; EU Horizon 2020 project VEO. Grant Number: 874735 NOW Stevin prize (Koopmans) Department of Medical Microbiology and Infection Prevention of the Amsterdam UMC