It is often difficult to make a definitive diagnosis of papillary breast lesions using core needle biopsy (CNB) specimens. We studied loss of heterozygosity (LOH) on chromosome 16q in order to assess its diagnostic use for papillary breast lesions in CNB specimens. Of 25 patients with intraductal papillary breast tumors, we extracted DNA from paired samples of tumor cells from CNB specimens and non-tumor cells from subsequent excision specimens and analyzed LOH at the D16S419 and D16S514 loci on chromosome 16q. LOH analysis results were compared with final diagnoses based on pathological features of the resected specimens. On the CNB specimens, 21 tumors were histologically diagnosed as indeterminate or suspicious for malignancy, while four tumors were unambiguously malignant. Of the 21 indeterminate or suspicious tumors, 11 were finally diagnosed as benign and ten as malignant, and on these, LOH analyses were informative for 8 of the 11 benign tumors and 7 of the 10 malignant tumors. LOH was also informative on two of the four tumors unambiguously malignant on CNB. None of the eight informative benign tumors showed LOH on 16q. Six of the eleven informative malignant tumors showed LOH on 16q. LOH on 16q was significantly different between CNB specimens of benign and malignant intraductal papillary tumors (P = 0.007). Analysis of LOH on 16q may be helpful in making a definitive diagnosis in cases of papillary breast lesions, in both excised and CNB specimens.