Here we investigated the pathogenesis and contact transmission of bovine- and human-derived highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype B3.13 viruses in mammalian models. Using reverse genetics, we rescued three naturally occurring viruses, rTX2/24 (bovine-derived), rTexas/37, and rMichigan/90 (both human-derived), and compared their infection dynamics, replication, and pathogenicity with the wild-type bovine TX2/24 strain in vitro and in vivo. All four viruses demonstrated comparable replication kinetics in four mammalian cell lines. However, the rMichigan/90 strain exhibited smaller plaques in bovine and human cells. In vivo studies showed that mice infected with any of the viruses succumbed to infection within 4–5 days; however, mice infected with the rMichigan/90 virus exhibited slightly lower viral replication and shedding compared with the other strains. The hamster model showed a milder infection phenotype than mice in which all viruses induced body weight loss and oral shedding, with robust virus replication observed in tissues, but the rMichigan/90 virus presented reduced replication and shedding. Contact transmission studies in hamsters revealed limited transmissibility for these viruses, with only one out of four animals inoculated with the rMichigan/90 virus transmitting it to a na?ve contact. These findings indicate that both bovine- and human-derived H5N1 genotype B3.13 viruses present high pathogenicity in rodent models, though the overall respiratory transmissibility remains low in the hamster model.