Seasonal influenza A viruses evolve rapidly, yet the emergence and molecular basis of resistance to the polymerase acidic (PA) inhibitor baloxavir marboxil (BXM), which is widely used in China, remain elusive. To address this, 3938 PA gene sequences were collected from influenza patients across mainland China between 2018 and 2025, from the national surveillance network and GISAID. By screening post-market mutations in the N-terminal domain of PA (PAN) that appeared in at least two samples and at a frequency below 50%, twenty-five single-point mutations were identified and additionally six linked mutations potentially associated with drug pressure. The impact of these mutations on BXM sensitivity was subsequently evaluated. Our analysis revealed the emergence of known mutations associated with reduced BXM sensitivity, including L28P, K34R, E198?K, although their prevalence remained low (2/3850, 0.05%). Notably, we identified a novel substitution, D27G, which conferred an approximately 12.4-fold reduction in BXM susceptibility compared with the wild-type virus and exhibited higher replication fitness than the canonical resistance mutation I38T, as demonstrated in human airway organoids. Molecular dynamics simulations further indicated that PA-D27G attenuates the interaction between PA and baloxavir acid, the active form of BXM. Epidemiological analysis showed that D27G mutation remained rare, being detected in four isolates (4/1247, 0.32%) in mainland China, and at a sporadic prevalence (<0.1%, 9/53132) across global isolates. In conclusion, these results demonstrate the early emergence of BXM-associated resistance in China and identify PA-D27G as a resistance-associated mutation with preserved viral fitness, underscoring the importance of continued genomic and epidemiologic surveillance.