The chromosomes of the tree shrew Tupaia belangeri exhibit highly localized CENP-B box sequences in the centromeric regions of most chromosomes. Telomeric sequences are present at the ends of all chromosomes and, in addition, at specific interstitial chromosomal sites that likely represent remnants of ancestral telomeres. This suggests that Robertsonian and tandem chromosome fusion events have occurred in the karyotypic evolution of Tupaiidae. In Tupaia skin fibroblasts CENP-B boxes are almost always clustered together at one pole of the interphase nucleus, whereas the telomeric domains are relatively evenly distributed throughout the whole nuclear volume. The observed orientation of the centromeres is reminiscent of the Rabl polarization of chromosomes; this is the first mammalian cell substrate in which such an higher-order chromosomal organization has been observed. CENP-B box sequences are found in several other mammalian species. The implications for recent parallel evolution of CENP-B binding motifs and concerted evolution of these sequences are discussed.