The variability of a small supernumerary marker chromosome (sSMC)-related phenotype is determined by the molecular component, the size, and shape of the marker chromosome. As fluorescence in situ hybridization has limitations regarding the resolution, efficiency, and accuracy. Recently, array comparative genomic hybridization (aCGH) was used for sSMC characterization. In this study, twenty cases with sSMCs were characterized by aCGH and FISH. Chromosomal origin of the marker chromosomes were successfully identified in seventeen of them. For the three cases with negative aCGH results, two of them were more likely due to that the sSMCs only contained centromere heterochromatin, whereas the reason for the remaining case with negative aCGH finding was uncertain. In order to establish a stronger genotype-phenotype correlation for clinical service in the future and avoid miss characterization, more sSMC cases were needed to be detailed characterized. This will help to clarify the variable clinical characteristics of sSMCs and provide additional information to aid clinical service and future research.