Objectives HCoV-HKU1 diversity and evolution was scarcely studied. We performed next-generation sequencing (NGS) and analysis of HCoV-HKU1 genomes over five years. Methods NGS used Illumina technology on NovaSeq 6000 following whole genome PCR amplification by a in-house set of primers designed using Gemi and PrimalScheme. Genome assembly and analyses used CLC Genomics, Mafft, BioEdit, Nextstrain, Nextclade, MEGA and iTol bioinformatic tools. Spike molecular modelling and dynamics simulations used Molegro Molecular Viewer/Hyperchem. Results Twenty-eight PCR systems allowed obtaining 158 HCoV-HKU1 genomes including 69 and 89 of genotype A and B, respectively. Both genotypes co-circulated during the study period but one predominated each year. 1,683 amino acid substitutions including 80 in ≥10 genomes were detected in genotype A relatively to a 2004 reference. H512R in spike, first detected in 2009 and reported as involved in antibody neutralization, was found in all genotype A, almost always with V387I and K478N, and was predicted here to significantly improve cellular TMPRSS2 protein binding. 1,802 amino acid substitutions including 64 in ≥10 genomes were detected in genotype B relatively to a 2005 reference. Conclusion This study substantially expands the global set of HCoV-HKU1 genomes. Genomics with protein structural analyses contributed to our understanding of HCoV-HKU1 evolution.