Background Amide proton transfer (APT) imaging has been increasingly applied in tumor characterization. However, its value in evaluating breast cancer remains undetermined. Purpose To assess the diagnostic performance of APT imaging in breast cancer and its association with prognostic histopathologic characteristics. Study Type Prospective. Subjects Eighty‐four patients with breast lesions. Field Strength/Sequence A 3.0 T/single‐shot fast spin echo APT imaging. Assessment APTw signal in breast lesion was quantified. Lesion malignancy, T stage, grades, Ki‐67 index, molecular biomarkers (estrogen receptor [ER] expression, progesterone receptor [PR] expression, human epidermal growth factor receptor [HER‐2] expression), molecular subtypes (luminal A, luminal B, triple negative, and HER‐2 enriched) were determined. Statistical Tests Student t ‐test, one‐way analysis of variance, receiver operating characteristic analysis, and Pearson's correlation with P < 0.05 as statistical significance. Results APTw signal was significantly higher in malignant lesions (1.55% ± 1.24%) than in benign lesions (0.54% ± 1.13%), and in grade III lesions than in grade II lesions (1.65% ± 0.84% vs. 0.96% ± 0.96%), and in T2‐ (1.57% ± 0.64%) and T3‐stage lesions (1.54% ± 0.63%) than in T1‐stage lesions (0.81% ± 0.64%) for invasive breast carcinoma of no special type. APTw signal significantly correlated with Ki‐67 index ( r = 0.364) but showed no significant difference in groups of ER ( P = 0.069), PR ( P = 0.069), HER‐2 ( P = 0.961), and among molecular subtypes ( P = 0.073). Data Conclusion APT imaging shows potential in differentiating breast lesion malignancy and associates with prognosis‐related tumor grade, T stage, and proliferative activity. Evidence Level 2 Technical Efficacy Stage 2