Cri-du-chat syndrome is associated with a deletion of the short arm of chromosome 5. Through the phenotypic and molecular analyses of individuals with a subset of the features associated with the syndrome, the genes involved in the syndrome have been mapped to two distinct critical regions. Deletion of a critical region in 5p15.2 results in the distinct facial features associated with the syndrome as well as the severe mental and developmental delay, while a deletion of 5p15.3 is associated only with the characteristic cat-like cry, the key diagnostic feature of the syndrome. Therefore, subtle differences in the extent of the 5p deletion can have a profound affect on the prognosis of the patient. In order to more easily differentiate between deletions that lead to the cri-du-chat syndrome phenotype and deletions that lead only to the isolated cat-like cry, we have constructed YAC contigs that span both critical regions. The YAC clones have been used to isolate cosmids mapping to each critical region and cosmids that lie just within the two critical region boundaries have been identified. We report here on the use of these cosmids as probes for fluorescent in situ hybridization experiments on interphase nuclei as a means of more accurately differentiating between small 5p deletions that coincide with a complete cri-du-chat syndrome phenotype and the severe mental and developmental delay that is associated with it and deletions that only delete the distal critical region that coincide with the isolated cat-like cry and a much improved prognosis.