F-alfatide II has been proven to have excellent clinical translational potential. In this study, we investigated F-alfatide II for identifying breast cancer and compared the performances betweenF-alfatide II and F-FDG. Forty-four female patients with suspected primary breast cancer were recruited. PET/CT images using F-alfatide II andF-FDG were acquired within 7 d. Tracer uptake in breast lesions was evaluated by visual analysis, and semiquantitative analysis with SUV and SUV Forty-two breast cancer lesions and 11 benign breast lesions were confirmed by histopathology in 44 patients. BothF-alfatide II and F-FDG had higher uptake in breast cancer lesions than in benign breast lesions ( < 0.05 for F-alfatide II, < 0.05 for F-FDG). The area under the curve ofF-alfatide II was slightly less than that of F-FDG. BothF-alfatide II and F-FDG had high sensitivity (88.1% vs. 90.5%), high positive predictive value (88.1% vs. 88.4%), moderate specificity (54.5% vs. 54.5%), and moderate negative predictive value (54.5% vs. 60.0%) for differentiating breast cancer from benign breast lesions. By combiningF-alfatide II and F-FDG, the sensitivity and negative predictive value significantly increased to 97.6% and 85.7%, respectively, with positive predictive value slightly increased to 89.1% and no change to the specificity (54.5%). The uptake ofF-alfatide II (SUV: 3.77 ± 1.78) was significantly lower than that of F-FDG (SUV: 7.37 ± 4.48) in breast cancer lesions ( < 0.05). F-alfatide II uptake in triple-negative subtype was significantly lower than that in luminal A and luminal B subtypes. By contrast, human epidermal growth factor receptor-2 (HER-2)-overexpressing subtype had higherF-FDG uptake than the other 3 subtypes. There were 8 breast cancer lesions with higher F-alfatide II uptake thanF-FDG uptake, which all had a common characteristic that HER-2 expression was negative and estrogen receptor expression was strongly positive. F-alfatide II is suitable for clinical use in breast cancer patients.F-alfatide II is of good performance, but not superior to F-FDG in identifying breast cancer.F-alfatide II may have superiority to F-FDG in detecting breast cancer with strongly positive estrogen receptor expression and negative HER-2 expression.