The development, incidence, and metastasis of tumors are all intimately correlated with 5-methylcytosine (m5C). However, uncertainty surrounds the function of m5C in acute myeloid leukemia (AML). In this study, multicenter AML data were collected and analyzed comprehensively to grasp the gene expression level, clinicopathological characteristics, prognostic significance of m5C in AML and its relationship with the tumor microenvironment (TME). The m5C gene-mediated scoring system (m5CSS) was created using principal component analysis, and multiple cox regression analyses were utilized to determine the prognostic relevance of the m5C score. The investigation of the correlation among m5C, immune characteristics, clinical characteristics, immune infiltration level, as well as drug reaction at immune checkpoints, and immunotherapy efficacy confirmed that the change of the characteristics of immune cell infiltration and patient prognosis are linked with the m5C gene. Moreover, the m5CSS was employed to assess the pattern of m5C modification. Further analyses showed that the m5C score can served as a reliable indicator of AML prognosis. Crucially, the prognostic value of the m5C score was validated in terms of drug resistance and immunotherapy. This work reveals that AML diversity and the generation of complex TMEs are both impacted by m5C modifications. Therefore, understanding the m5C modification pattern will improve grasp of TME infiltration characteristics and assist exploring more efficient immunotherapeutic approaches.