Array comparative genomic hybridization (aCGH) is a technique enabling high-resolution, genome-wide screening of segmental genomic copy number variations (CNVs). It is becoming an essential and a routine clinical diagnostic tool and is gradually replacing cytogenetic methods. Most of the clinically available aCGH platforms are designed to detect aneuploidies, well-characterized microdeletion/microduplication syndromes and subtelomeric or other unbalanced chromosomal rearrangements. In addition, aCGH can uncover numerous CNVs of unclear significance scattered throughout the human genome. But this technology is not able to identify balanced chromosomal imbalances such as translocations and inversions and some ploidies. aCGH increased the ability to detect segmental genomic CNVs in patients with global developmental delay, mental retardation, autism, multiple congenital anomalies and dysmorphism, and is becoming a powerful tool in disease gene discovery and prenatal diagnostics. This tool is also showing promising data in cancer research and in the diagnosis, classification and prognostification of different malignancies.