Chromosomal instability (CIN) is a common but not universal feature of colorectal cancer (CRC); however, the molecular basis for CIN is controversial and poorly understood. There are many plausible mechanisms proposed for CIN, including disruption of G1/S and G2/M checkpoint regulation, and alterations in the spindle checkpoint genes. However, mutations in individual growth regulatory genes are not commonly observed in CRC. Therefore, a more comprehensive analysis of the genes involved in each cell cycle checkpoint regulatory pathway might be required to evaluate a possible role for involvement in CIN. We investigated the presence of high copy amplification of the cyclin E, Aurora-A, Skp2 genes, mutation of ubiquitin ligase CDC4, and promoter methylation of Mad2L1, as well as the expression of the gene products in a panel of 11 human CRC cell lines as well as 48 human CRC specimens. In the cell lines with CIN, we found amplification of the Aurora-A, cyclin E and Skp2 genes, and a mutation in the CDC4 gene, all of which resulted in altered expression of the cognate proteins. In the human CRC tissues, amplification of Aurora-A was frequent (29%), while alterations were rarely observed in cyclin E, Skp2 or CDC4. Aurora-A amplification was strongly associated with a high fractional allelic loss score (p = 0.0001), but not with microsatellite instability, nor with the promoter methylation phenotype in these tumors. Our data confirm involvement in the CDC4-cyclin E pathway of the development of the CIN phenotype in human CRC, and find that amplification of the Aurora-A is a common target for disruption of this pathway.