A five-year-old Caucasian male presented with developmental delay, minor dysmorphic features, and hyperactivity. Cytogenetic analysis showed the presence of a marker chromosome in the majority of cells analyzed. Fluorescence in situ hybridization (FISH) analyses using several alpha satellite probes, including D13Z1/D21Z1, did not reveal any signal on the marker chromosome. Subsequent multicolor FISH (M-FISH) indicated the marker to be derived from chromosome 13, and FISH with a chromosome 13 paint confirmed this finding. The absence of D13Z1/D21Z1 signal on the marker suggested that it was analphoid in nature. Comparative genomic hybridization (CGH) was utilized to further characterize the region of chromosome 13 from which the marker originated, and unexpectedly revealed a gain of chromosomal material at both the centromeric regions of chromosomes 3 and 13. In view of the CGH results, extensive FISH studies with D3Z1 and D13Z1/D21Z1 were performed and revealed the presence of four cell lines comprising one normal cell line (50.5%), a cell line with a chromosome 3 derived marker (19%), a cell line containing a marker derived from chromosome 13 (20%), and a cell line with both markers (10.5%). As the two markers appeared morphologically similar by GTG banding, all 47,XY metaphases in the initial analysis were thought to comprise only a single marker. This is the first report, to our knowledge, of the presence of a chromosome 3 and a chromosome 13 marker in mosaic condition in a congenital disorder. In light of our experience, we urge caution in interpreting karyotypes with marker chromosomes. Our case illustrates the limitations of fluorescent DNA probes and sampling errors.