BACKGROUND: Differentiating molecular subtypes and identifying biological markers in breast cancer (BC) are essential for prognostic stratification and treatment selection. This study aimed to compare the effectiveness of amide proton transfer-weighted imaging (APTWI) and diffusion-weighted imaging (DWI) in differentiating molecular subtypes and predicting the biological status of BC. METHODS: This retrospective study included 109 women (aged 50.8±10.8 years) with BC who underwent 3T APTWI and DWI between May 2023 and January 2024. Patients were categorized by molecular subtypes and expression levels of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67. Magnetization transfer ratio asymmetry (MTRasym) and apparent diffusion coefficient (ADC) values were measured. The area under the receiver operating characteristic (ROC) curve (AUC) was used to assess the performance of MTRasym and ADC values in distinguishing subtypes. Pearson's correlation analysis was used to examine the relationship between MTRasym, ADC values, and the Ki-67 proliferation index. RESULTS: Triple-negative (TN) cancers (3.03%±0.56%) had significantly higher MTRasym values than luminal A (2.25%±1.00%) and luminal B (2.39%±0.81%) cancers (P=0.006, 0.012). HER2-enriched cancers (2.93%±0.71%) also had significantly higher MTRasym than luminal A cancers (P=0.039). MTRasym and ADC values were significantly higher in ER-negative (ER-) than they were in ER-positive (ER+) cancers (P<0.001, P=0.040), and MTRasym values were higher in PR-negative (PR-) and high-Ki-67 cancers (P<0.001, P=0.013). AUC values for MTRasym ranged from 0.699 to 0.799, depending on the subtype and biological marker comparison. MTRasym and ADC values showed a weak positive correlation with the Ki-67 index (r=0.37, P<0.001, and r=0.31, P=0.003). CONCLUSIONS: APTWI is more effective than DWI for differentiating BC subtypes and predicting biological markers, providing valuable insights for clinical management.