Introduction: The Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly syndrome characterized by a distinctive facial appearance, prenatal and postnatal growth deficiency, psychomotor delay, behavioural problems, and malformations of the upper extremities. Recently mutations in NIPBL, the human homolog of the Drosophila Nipped-B gene, were found to cause CdLS. In total mutations have been found in 39% of reported cases. Methods: We investigated 39 CdLS patients in the Netherlands both clinically, through 3D scanning, behaviourally, and molecularly. Results: We report mutations in 56% of cases after personal clinical examination of each patient before inclusion. Truncating mutations were found to cause in general a more severe phenotype but this correlation was not absolute. Three-D face imaging demonstrated the potential for classifying facial features. Behavioural problems were highly correlated with the level of adaptive functioning, and also included autism. No correlation of behaviour with the type of mutation was found. Discussion: In this study of a major proportion of the CdLS population in a single country, a mutation could be found in 56% of patients who received careful clinical investigation. Although truncating mutations were found to be causing in general a more severe phenotype, this correlation was not absolute and it seems likely that other genetic or environmental factors influence the phenotype as well. Pronounced behavioural problems including autism were found more often in patients with truncating mutations but similar problems did occur in missense mutations as well.