Since 2020, H5N1 highly pathogenic avian influenza (HPAI) viruses of clade 2.3.4.4b have been rapidly spreading in wild birds but have also caused a large number of mammalian infections and more than 70 known human cases. Importantly, this H5N1 clade has also crossed the species barrier into dairy cattle in the US in late 2023/early 2024. The neuraminidase (NA) protein of the N1 subtype can feature truncations in its stalk domain, which have been identified as putative virulence factors in poultry but seem to have a negative impact on transmission in mammals. Since its emergence, the vast majority of HPAI H5N1 A/goose/Guangdong/1/1996-lineage isolates have featured this truncated version of the NA stalk domain. Here, we report that this changed with the 2020 expansion of clade 2.3.4.4b H5N1 and that the majority of isolates-including the strains circulating in dairy cattle-feature a long NA stalk domain.IMPORTANCEWhile the truncated version of the N1 neuraminidase stalk domain may be associated with increased virulence in poultry, the long version of the stalk domain has been associated with increased transmissibility in mammals. The vast majority of highly pathogenic H5N1 of clade 2.3.4.4b that is currently circulating globally features the long stalk version of the neuraminidase, which may increase the risk for these viruses to become human-to-human transmissible.