The prognostic value of gene abnormalities in colorectal cancer is unclear, varies between investigators, and may be unreliable due to tumor heterogeneity. We analyzed tumors completely divided into biopsies to characterize the overall distribution of LOH at 5q, 17p, 18q, K-ras mutations, and methylation of the p16 and MGMT genes. Seventeen tumors (stage II-III) were completely divided into biopsy cubes from which DNA was sequentially analyzed. The results from fragment analysis for LOH at 5q, 17p, 18q, TGGE of the K-ras, and MSP for p16 and MGMT genes were used to characterize the occurrence of these aberrations. LOH at 18q, 17p, and LOH 5q were present in 17/17, 15/17, and 15/16 tumors, respectively. p16 and MGMT were methylated in 16/17 and 14/17, respectively. The frequency of these aberrations varied largely between tumors. K-ras-mutations, present in 8/17 tumors, were much more consistently distributed in mutated tumor. Methylation of p16 and MGMT and LOH at 18q, 17p, and 5q are present within the tumor mass in a large majority of CRC tumors and are of no or little prognostic value. K-ras mutations appear more homogeneously distributed. This has clinical relevance for biopsing to predict anti-EGFR response.