Breast cancer is often detected as multiple lesions clinically and/or histopathologically. To examine if the origin of such lesions can be identified objectively by comparison of their loss of heterozygosity (LOH) patterns, LOH on chromosome 16q was analyzed in a total of 60 cases of multiple breast cancer by Southern blot analysis. Based on continuity among tumors and satellite nodule features, 30 cases of unilateral multiple cancer were classified morphologically into 3 groups: A, multicentric origin (11 cases); B, multifocal invasion of one intraductal carcinoma (15 cases); and C, intramammary metastases (4 cases). As controls, group D, synchronously bilateral breast cancers (11 cases), and group E, sets of a primary tumor and a lymph node metastasis (19 cases), were also examined. On a highly probable assumption that LOH on 16q occurs randomly in 50% of breast cancer cases at an early stage, the number of cases showing a concordant LOH pattern on 16q among tumors was compared between observed data, and the value was estimated from a normal distribution model in each group. In groups A and D, the allele pattern on 16q among tumors was concordant in 5 of 11 cases each, thus supporting their independent occurrence and multicentric origin, whereas the LOH pattern among tumors was identical in all of the cases in groups B, C, and E, thus supporting their monocentric origin. This comparison of the LOH pattern in multiple breast cancer was shown to yield results compatible with the morphological classification and was suggested to be of diagnostic value.