Identifying marker chromosomes of unknown origin in the clinical cytogenetics laboratory has been a problem historically, despite advances in specialized staining techniques. Determination of the origin of these marker chromosomes in patients with congenital or malignant neoplastic disorders is essential for more complete diagnosis, counseling, and treatment. The authors describe the use of fluorescent in situ hybridization with chromosome-specific alpha-satellite DNA probes to identify the origin of marker chromosomes in two patients with congenital disorders and three patients with malignant neoplastic disorders. The impact of firm identification of the marker chromosome for the diagnosis of these patients is discussed. The authors also discuss the feasibility of using this technique routinely in the clinical cytogenetics laboratory.