We have tested the tandem fusion hypothesis of the origin of the Indian muntjac karyotype (2n = 6/7) by using reciprocal chromosome painting between the Indian muntjac, Chinese muntjac (n = 46) and brown brocket deer (2n = 70 + 3B) with chromosome-specific paint probes derived from flow-sorted chromosomes of these three deer species. Our results have shown that the euchromatic blocks of all chromosome arms of the brown brocket deer have been conserved apparently unchanged in number and content in the Indian muntjac. While confirming the conservation in toto of most of Chinese muntjac euchromatin in the karyotype of the Indian muntjac, we demonstrate that the synteny of chromosomes 1, 2, 3, 4 and 5 of the Chinese muntjac has been disrupted by chromosome rearrangements other than fusions. This indicates that the present karyotype of the Indian muntjac cannot be reconstructed from the hypothetical Chinese muntjac-like 2n = 46 ancestral karyotype exclusively by chromosome fusions. Furthermore, we have shown that the breakpoints of these rearrangements appear to have occurred near to the fusion points formed during the origin of the 2n = 46 karyotype of the Chinese muntjac from a 2n = 70 karyotype, which is believed to be ancestral for the family Cervidae. Moreover, we substantiate that on the Indian muntjac chromosomes, the C5 probe, which is derived from the centromeric satellite sequences of the Chinese muntjac, maps to the putative fusion points determined by comparative chromosome painting and presumably represents the remnants of ancestral centromeric sequences.