Aim: To evaluate the usefulness of localization needles under mammographic, ultrasound or fiberoptic ductoscopy guidance for non-palpable breast lesions. Patients and Methods: Eighty-three patients undergoing needle localization and biopsy of non-palpable breast lesions under mammographic, ultrasound or fiberoptic ductoscopy guidance from June 2013 to December 2014 in Beijing Friendship Hospital were included in the study. The preoperative imaging assessment, application of localization needles, surgical operation and pathological examination were recorded and analyzed retrospectively. Results: A total of 83 localization and biopsies were carried out, of which 27 were performed under mammographic guidance, 32 under ultrasound guidance and 24 under fiberoptic ductoscopy guidance. Twenty-seven cases of breast microcalcifications were localized under mammographic guidance and surgically removed, of which eight cases were pathologically diagnosed as malignant. Thirty-two cases of non-palpable breast lesions were localized under ultrasound guidance and 30 pathologically diagnosed, of these, four cases were pathologically diagnosed as malignant. Twenty-four cases of intraductal space-occupying lesions were localized under ductoscopy guidance and surgically removed, of which five cases were pathologically diagnosed as malignant. Conclusion: Utilization of localization needles under mammographic, ultrasound or fiberoptic ductoscopy guidance for non-palpable breast lesions is a safe and effective procedure, and is helpful in the diagnosis of breast cancer. With the help of this procedure, more malignant lesions can be localized and surgically removed.