Intraoperative ultrasound (IOUS) can be used in the operation theatre for localization of non-palpable breast cancers. In this prospective cohort study, we compared the yield of IOUS to guidewire localization (GWL). A total of 258 consecutive patients with non-palpable invasive breast cancer underwent breast conserving surgery between 1999 and 2010. GWL was performed in 138 (54 %) and IOUS in 120 (46 %) patients. Tumor dimensions, resection volume, margin status and re-excision rates were compared by means of multivariate regression analysis. The groups were similar in terms of age, histological subtype and presence of DCIS. Lesions in the IOUS group were larger (1.24 vs. 0.98 cm, P < 0.001), while microcalcifications were more common in the GWL group (19 vs. 3 %, P < 0.001). Even after stratification for tumor diameter, presence of DCIS and findings on mammography, resection volumes were similar in both groups. Tumor-free resection margins were obtained in >93 % of patients (93.5 % with GWL vs. 93.3 % with IOUS, P = 0.958) and re-excision was performed in 11 % of patients undergoing GWL and 12.5 % of patients undergoing IOUS (P = 0.684). For localization of non-palpable breast cancer, IOUS is a reliable alternative to GWL, as it achieves similar results in terms of complete tumor removal, re-excision rate and excised volume.