A variant sequence of minor satellite that exhibits a polymorphism in strains and a subspecies of the mouse by pulsed-field gel analysis has been cloned. A segregation study using intrasubspecific backcross mice reveals that the variant maps to the centromeric region of chromosome 2. A long-range physical analysis of the region shows that the variant family is probably located between the proximal telomere and the major satellite. The sequence probe provides an important marker of the chromosome 2-specific centromere.