Despite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. In many cases, the classification of these lesions as either two separate primary tumors or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. To establish the origin of tumors, we assessed tumor cell clonality using PCR-based microsatellite analysis on microdissected archival tissues for loss of heterozygosity (LOH) and microsatellite instability (MSI) in a series of 19 paired synchronous and metachronous tumors from several organs. As a control group, 15 autopsy cases with an unequivocally recognizable primary tumor and associated metastases were also examined. Based on LOH and MSI findings, and using a panel of 4 to 12 (median 7) microsatellite markers, we were able to establish the clonal pattern of microsatellite changes in 17 out of 19 (89%) biopsy cases and thus determine if they were either double primary tumors (41%) or metastases (59%). Of interest, identical or similar pattern of microsatellite abnormalities were detected in 15 primary tumors and corresponding metastasis from autopsies. Our results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms and metastases in synchronous and metachronous tumors.