Small supernumerary marker chromosomes (sSMCs) are often associated with developmental abnormalities and malformations are de novo in approximately 60% of the cases. Fluorescence in situ hybridization (FISH) techniques using various probes provided the possibility to analyze and characterize sSMCs, which is highly important for prenatal diagnosis and genetic counseling. We now present the establishment of a specific strategy to identify the origin and structure of the sSMCs using a combination of conventional banding and classical FISH techniques. Based on this strategy, in a series of 15,792 prenatal karyotypes, 20 cases with sSMCs (prevalence 1.26 per 1000) were diagnosed. Eighteen of these cases were completely analyzed by FISH using commercial probes and Chromoprobe Multiprobe-I System. Out of 20 sSMCs 12 were satellited (10 bisatellited and two monosatellited) (60%) and eight were non-satellited (six ring-like and two isochromosomes) (40%). sSMCs were mostly derived from chromosome 15 (10/20) (50%). Euchromatin material was found in 13 cases by various banding and FISH techniques, while in six of 20 sSMCs there was no evidence of euchromatin material. Parental karyotypes could be evaluated in 15 cases and familial inheritance was found in only three of them (20%). We conclude that the proposed strategy for the identification and characterization of sSMCs is accurate and represents a good alternative to novel FISH techniques for modestly equipped cytogenetic laboratories.