We have hybridized all 28 chromosome-specific painting probes from the domestic sheep (Ovis aries, 2n = 54) onto metaphase chromosomes of the Indian muntjac deer (Muntiacus muntjak vaginalis, 2n = 6,7) and identified 35 conserved chromosomal segments. Results from this study show that most of the sheep acrocentric chromosomes hybridized to single regions in the Indian muntjac genome. This conserved hybridization pattern supports the concept that the large Indian muntjac chromosomes were derived from multiple tandem fusions from an ancestral deer species. Using previously reported fluorescence in situ hybridization data in which human chromosomes were hybridized onto the Indian muntjac genome, we were able to align chromosomal segments of the sheep and human genomes. Using this three-species genome alignment approach, we have identified a minimum of 42 conserved chromosomal segments between sheep and human genomes including 7 new regions not previously reported.