Plant and animal karyotypes sometimes contain variable elements, that are referred to as additional or B-chromosomes. It is generally believed that B-chromosomes lack major genes and represent parasitic and selfish elements of a genome. Here we report, for the first time, the localization of a gene to B-chromosomes of mammals: red fox (Vulpes vulpes) and two subspecies of raccoon dog (Nyctereutes procyonoides). Identification of the proto-oncogene C-KIT on B-chromosomes of two Canidae species that diverged from a common ancestor more than 12.5 million years ago argues against the current view of B-chromosomes. Analyses of fox B-chromosomal C-KIT gene from a flow-sorted fox B-chromosome-specific library revealed the presence of intron-exon boundaries and high identity between sequenced regions of canine and fox B-chromosomal C-KIT copies. Identification of C-KIT gene on all B-chromosomes of two canid species provides new insight into the origin and evolution of supernumeraries and their potential role in the genome.