Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.