Abstract: Molecular imaging, using positron emission tomography (PET), has become an integral step in the evaluation of many patients with malignancy. However, its use in patients with breast cancer has been limited by the lower levels of 18F-fluorodeoxyglucose (FDG) uptake in some breast malignancies compared to other cancers, the small size of many breast cancers, and the need for biopsy under PET guidance. High-resolution breast PET, or positron emission mammography (PEM), with biopsy guidance software, now addresses these issues. We report a prospective, multicenter study designed to test the efficacy and safety of PEM biopsy guidance software in women with FDG-avid breast lesions worrisome for malignancy. The intervention chosen was vacuum-assisted core biopsy. Nineteen subjects underwent a total of 24 PEM-guided biopsies. All lesions were successfully targeted and sampled as determined by post-biopsy image scan evaluation, specimen imaging, and pathologic concordance. Invasive cancer was identified in 13 of 24 lesions (54%), while four (17%) were high-risk lesions and three of these were upgraded to malignancy at excision. No serious adverse events occurred and all patients found the procedure to cause only minimal to mild discomfort. High-resolution PEM-guided breast biopsy is both safe and effective for the sampling of PET-depicted breast lesions.