Basal cell carcinoma (BCC) is the most common skin tumor in Caucasians. Loss of heterozygosity (LOH) of chromosome 9 (9q22.3) is common in BCCs, and indirectly implies their monoclonal origin. Although BCCs are considered monoclonal tumors, the clonal origin of anatomically distinct tumors in the same patient was rarely investigated. Forty-one anatomically distinct BCCs from 15 female patients were genotyped for LOH at chromosome 9q22.3, and the inactivation pattern of X-chromosome using the androgen receptor polymorphic site. Overall, 11 women with 30 tumors were heterozygous (informative) with either one of the two 9q markers or the androgen receptor polymorphism. LOH of 9q was detected in 20 tumors from seven patients, and in all tumors derived from the same patient, the same allele was lost. An identical X-chromosome inactivation pattern was noted in all four tumors taken from one patient, and in another informative patient, one tumor demonstrated LOH and the other retained it. In five tumors from two patients, there was no evidence of monoclonality using either technique. We conclude that the majority of anatomically distinct BCCs are monoclonal neoplasms and may represent expansion of the same clone.