The global spread of the A/goose/Guangdong/1/96-lineage H5N1 highly pathogenic avian influenza (HPAI) viruses is accompanied by an expanded host range and the establishment of sustained viral transmission among dairy cattle. To evaluate if the evolving H5N1 viruses have changed tissue tropism over time, we compared the binding patterns of recombinant hemagglutinin (HA) proteins derived from clade 1 (A/Vietnam/1203/04, H5VN) and circulating clade 2.3.4.4b viruses detected from a wild bird (A/Eurasian Teal/Hong Kong/AFCD-HKU-23-14009-01020/2023, H5HK) and dairy cattle (A/bovine/Ohio/B24OSU-439/2024, H5OH). The HA protein of A(H1N1)pdm09 virus was included for comparison. Using bio-layer interferometry, H1 protein preferentially bound to the 2,6-linked sialoside 6´SLNLN while H5 proteins preferentially bound to the 2,3-linked sialoside 3´SLN. H5OH showed higher binding affinity to 3´SLN than H5HK and H5VN. The attachment pattern of H1 and H5 proteins to the respiratory tissues of different species and dairy cattle mammary glands were evaluated. Compared to the H1 protein, H5 proteins showed stronger binding to the lung epithelial cells of cat, cattle, chicken, ferret, human, and pig, and the clade 2.3.4.4b H5 proteins exhibited increased binding to pig and cattle bronchial epithelial cells. All H5 proteins attached to the alveolar and cistern epithelial cells in mammary glands where α2,3-linked and α2,6-linked sialyl glycans were detected by Maackia amurensis lectin II and Sambucus Nigra Lectin, respectively. Taken together, the HA proteins of clade 1 and 2.3.4.4b H5N1 viruses generally share comparable attachment patterns to avian and mammalian tissues, despite of evolving into antigenically distinct clades over the past 3 decades.