Objective To investigate the clinical significance of the proportion of S100A9-high-expressing cells in the malignant cell population of acute myeloid leukemia （AML）， and to analyze its association with chemotherapeutic response and patient prognosis， so as to provide evidence for the identification and prognostic evaluation of high-risk AML patients. Methods Survival curves were initially generated using the BeatAML and TCGA datasets based on S100A9 transcript expression levels. Subsequently， a total of 80 patients with AML， consisting of 44 chemotherapy-sensitive cases and 36 chemotherapy-resistant cases， along with 10 healthy donors， were recruited. Multiparameter flow cytometry was carried out to assess the proportion of S100A9-high-expressing cells within the pre-defined AML malignant cell population. The relationships between this proportion and post-chemotherapy response as well as survival outcomes were analyzed. Immunocytochemistry and immunohistochemistry were further employed for validation. Results Survival analysis based on the overall expression of S100A9 mRNA revealed no statistically significant difference in either the TCGA or BeatAML dataset. Nevertheless， the proportion of malignant cells with high S100A9 expression was significantly higher in non-remission AML patients compared to those who achieved remission. The area under the receiver operating characteristic （ROC） curve was 0.765， and the optimal cutoff value was determined to be 12.65%. Patients characterized by a high proportion of malignant cells with high S100A9 expression， as defined by this cutoff， exhibited poorer survival outcomes and an unfavorable prognosis. Conclusions The proportion of S100A9-high-expressing malignant cells in AML could serve as a potential prognostic indicator. In comparison to the overall S100A9 expression， this parameter might be more effective in identifying high-risk AML patients.