Baloxavir marboxil, a cap-dependent endonuclease inhibitor, was approved in Japan in 2018 for the treatment and prophylaxis of influenza. Its active form, baloxavir acid, binds to the polymerase acidic (PA) protein endonuclease domain, inhibiting viral RNA cleavage. PA substitutions (e.g., E23K, I38T, E199G) have been associated with reduced susceptibility to baloxavir. During nationwide monitoring in Japan, we identified influenza A(H1N1)pdm09 and A(H3N2) viruses carrying a PA E199K substitution. Database analysis revealed that PA E199K is rare, detected in only 0.01% of A(H1N1)pdm09 and A(H3N2) viruses. Because its impact on baloxavir susceptibility has not been reported, here, we characterized PA E199K mutant viruses in vitro. Phenotypic analysis showed a 5.0–5.2-fold increase in baloxavir EC50 values in PA E199K mutants, indicating reduced baloxavir susceptibility similar to PA E199G. However, replication efficiency of PA E199K mutants was significantly lower than wild-type viruses, suggesting impaired viral fitness. Unlike PA E199G, PA E199K introduces charge and steric changes that may further reduce replication capacity. While PA E199G mutants have led to a community cluster, PA E199K has only been detected sporadically, likely due to its greater impairment of viral replication. The PA E199K mutants were susceptible to neuraminidase inhibitors. Given the increasing global use of baloxavir, continuous monitoring of resistance-associated substitutions is essential for public health and clinical management.