The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor alpha (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption of the equine synteny. Next, human (HSA) Chromosomes (Chrs) 16q and 4 specific paints, known to be homologous to ECA3p and 3q, respectively, were applied to detect homologous chromosomal segment(s) in donkey. Finally, four genes (MC1R, ALB, PDGFRA, KIT) and two equine microsatellite markers (SGCV18 and SGCV33) located on ECA3 were FISH mapped to donkey chromosomes. The findings refined the cross species painting homology results and added six new markers to the nascent donkey gene map. The hypothesis that Tobiano coat color in horses may be associated with a chromosomal inversion involving genes within LG2 was tested by G-banding-based cytogenetic analysis and ordering of four loci-KIT, PDGFRA, albumin (ALB), and MC1R-in Tobiano and non-tobiano (homozygous as well as heterozygous) horses. However, no difference either in banding patterns or location/relative order of the genes was observed in the three classes. The study highlights successful FISH mapping of BAC probes across evolutionarily diverged species, viz., pig and horse/donkey, and represents the first use of large-sized individual clones across distantly related farm animals.