Array-based comparative genomic hybridization (CGH) allows for the simultaneous examination of thousands of genomic loci at 1–2 Mb resolution. Copy number alterations detected by array-based CGH can aid in the identification and localization of cancer causing genes. Here we report the results of array-based CGH in a set of 125 primary colorectal tumors hybridized onto an array consisting of 2463 bacterial artificial chromosome clones. On average, 17.3% of the entire genome was altered in our samples (8.5 ± 6.7% gained and 8.8 ± 7.3% lost). Losses involving 8p, 17p, 18p or 18q occurred in 37, 46, 49 and 60% of cases, respectively. Gains involving 8q or 20q were observed 42 and 65% of the time, respectively. A transition from loss to gain occurred on chromosome 8 between 41 and 48 Mb, with 25% of cases demonstrating a gain of 8p11 (45–53 Mb). Chromosome 8 also contained four distinct loci demonstrating high-level amplifications, centering at 44.9, 60, 92.7 and 144.7 Mb. On 20q multiple high-level amplifications were observed, centering at 32.3, 37.8, 45.4, 54.7, 59.4 and 65 Mb. Few differences in DNA copy number alterations were associated with tumor stage, location, age and sex of the patient. Microsatellite stable and unstable (MSI-H) tumors differed significantly with respect to the frequency of alterations (20 versus 5%, respectively, P < 0.01). Interestingly, MSI-H tumors were also observed to have DNA copy number alterations, most commonly involving 8q. This high-resolution analysis of DNA copy number alterations in colorectal cancer by array-based CGH allowed for the identification of many small, previously uncharacterized, genomic regions, such as on chromosomes 8 and 20. Array-based CGH was also able to identify DNA copy number changes in MSI-H tumors.