Two synchronously arising primary squamous cell carcinomas (SCC) originating from separate sites in the anterior floor of mouth (FOM) and the pyriform sinus (PS) were evaluated by karyotype and fluorescence in situ hybridization (FISH) to determine whether they were of common or independent ancestry. The primary tumors were designated Henry Ford Hospital (HFH)-SCC-8a (FOM) and HFH-SCC-9a (PS), and the respective recurrent tumors after chemotherapy and radiation were designated -8b and -9b. HFH-SCC-8a and -8b were cultured and had closely related hypotetraploid karyotypes of monoclonal origin. Karyotypes could not be obtained from the second primary tumor HFH-SCC-9a or its recurrence -9b. However, we used karyotypes from HFH-SCC-8a and -8b as a guide to select FISH probes for the histological evaluation of genetic markers in tumor sections. Fluorescence in situ hybridization on metaphase chromosomes from the cell cultures was useful in modifying the tumor karyotypes. Fluorescence in situ hybridization identified a chromosome Y rearrangement that was not obvious from the HFH-SCC-8a and -8b karyotypes, and this Y rearrangement served as a unique clonal marker. Using two probes for the Y chromosome we showed that all four tumors shared the same Y rearrangement with loss of Yq (DYZ1) and retention of Ycen (DYZ3). Furthermore, FISH showed that all four tumors had the same aneuploidy patterns for chromosomes X, Y, 7, 9, 15, 16, and 17. From karyotypic and FISH analysis disomy for X and 9 centromere regions and the rearranged Y were all predicted and observed in the tumor tissue sections. Tetrasomy and trisomy for 7, 15, 16, and 17 were predicted from the karyotypes and this also was observed using FISH in all four tumors. These FISH aneuploidy patterns and the presence of a clonal Y marker in all four tumor samples indicate that the synchronous primaries and their recurrences were of monoclonal origin.