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In 2022/2023, Europe experienced its third consecutive season of high-pathogenicity avian influenza. During this period, the Czech Republic was again severely affected. For the first time, the number of culled birds approached one million, which was three times higher than in previous seasons. In parallel to the outbreaks in poultry, mass die-offs of gulls were also observed. In the present study, we performed whole-genome sequencing and phylogenetic analysis of 137 H5N1 strains collected in the Czech Republic in 2022/2023 (94.6% of all outbreaks or locations). The analysis revealed four distinct genotypes: AB, CH, BB and AF. Phylogenetic analysis suggested that the AF genotype persisted from the previous H5N1 season without reassortment. In addition, the genotype BB, which was detected mainly in gulls, showed a noticeable strain diversity at the local level. This virus was also responsible for a single outbreak in commercially bred turkeys. Finally, an interesting spatio-temporal cluster with three co-circulating H5N1 genotypes, AB, CH and AF, was identified with no evidence of intrasubtype reassortment. Highly sensitive molecular surveillance and the timely sharing of genomic sequences and associated metadata could greatly assist in tracking the spread and detecting molecular changes associated with the increased virulence of this potentially zoonotic pathogen. [ABSTRACT FROM AUTHOR]