Since October 2024, 55 human cases of influenza A(H5N1), clade 2.3.4.4b, were reported in the US. Sequencing of 46 viruses identified genotypes B3.13, D1.1, and D1.3. Genomes of viruses were analyzed for mutations associated with reduced antiviral susceptibility. Except for two, viruses from human cases were assessed as susceptible to antivirals and confirmed by in vitro susceptibility testing of representative viruses with M2 blockers, neuraminidase (NA) inhibitors, and the polymerase acidic (PA) inhibitor baloxavir. One D1.1 virus had an M2-S31N substitution, which conferred cross-resistance to M2 blockers. One B3.13 virus had a PA-I38M substitution and displayed 17-fold reduced baloxavir susceptibility in cell culture. In mice, baloxavir, given orally at 5, 15, and 45?mg/kg bid for 5 days starting at 2?hours post-infection, was notably less effective against the PA-I38M virus compared to the closely matched control virus, as shown by survival rate, time-to-death, weight loss, and viral replication in various organs. Replication of the PA-I38M virus was mildly attenuated in mice but not in cell culture. Viruses collected from animals also contained mutations associated with reduced antiviral susceptibility, including M2-S31N, PA-I38?T/M, and NA-H275Y, albeit at low frequency. Regardless of genotype, most mutations were found in NA. Using recombinant NA proteins generated from B3.13 and D1.1 backgrounds, we showed that among mutations that reduced susceptibility of NA inhibitors, only NA-H275Y had no effect on the enzyme’s function. Spontaneous emergence of drug-resistant influenza viruses, especially those that maintain replicative fitness, pose persistent threat to human health and must be closely monitored.