Although aneuploidy is commonly observed in human cancers, the molecular mechanism underlying aneuploidization remains unclear. We used multiploid cancer model that had diploid and aneuploid cancer cells within the same cancerous tissue and attempted to detect specific epigenetic alterations associated with tumor cell aneuploidy. Thirty-four multiploid colorectal cancers were subjected to crypt isolation and cell sorting, and paired diploid and aneuploid cancer cells were separated from each cancerous tissue. A methylated CpG island amplification provided a considerable number of CpG sequences that showed different methylation status between the above 2 cell populations. BLAST homology search revealed 24 different candidates (11 hypermethylated and 13 hypomethylated) from these sequences. The putative promoter sequence of the SALL4 (sal-like 4, a human homolog to Drosophila spalt) gene was particularly more frequently hypermethylated in aneuploid cells (62%) than diploid ones (35%) in the 34 multiploid cancers. Moreover, such hypermethylation occurred more often in aneuploid cancers (8 of 16, 50%) than diploid cancers (3 of 18, 17%). In combination with demethylation study on cultured cells, these results implied a possible association between epigenetic silencing of SALL4 and tumor cell aneuploidy. SALL4 may be one of important key players that act as "caretakers" for chromosomal stability. Our new approach is a powerful tool for the global identification of such key players.