A highly repetitive DNA clone, designated Rt-Pst3, was isolated from the PstI digest of Canadian woodland caribou (Rangifer tarandus caribou; 2n = 70) genomic DNA. It was found to be a 991 bp monomer of a tandemly repeated DNA sequence comprising about 5.7% of the genome and localized to the centromeric regions of all caribou acrocentric autosomes. Southern blot analyses revealed that this caribou satellite DNA sequence was well conserved in the genomes of five other deer species studied. In situ hybridization studies revealed Rt-Pst3-homologous DNA sequences in the centromeric regions of white-tailed deer chromosomes and Asian muntjac chromosomes, as well as at several interstitial chromosome regions in Indian muntjac chromosomes. Comparisons of the Rt-Pst3 DNA sequence to previously identified centromeric satellite DNA fragments from three other deer species revealed considerable DNA sequence similarity. The first ca. 800 bp of the Rt-Pst3 clone was found to share 73.8% similarity to the CCsatI clone of the European roe deer, 64.7% sequence similarity to the C5 DNA clone of the Chinese muntjac, and 64.8% and 65.6% sequence similarity to the 1A and B1 clones of the Indian muntjac, respectively. Moreover, the last 191 bp of the Rt-Pst3 clone was found to share about 60% DNA sequence similarity to the first 191 bp of the same clone. Amplification of one original ca. 800 bp monomer unit, along with the first 191 bp of the following juxtaposed monomer unit could have resulted in the tandemly repeated, 991 bp monomer unit now seen in the caribou genome. It is postulated that the centromeric satellite DNA found in other deer species, having repeat lengths greater than 800 bp, could also have evolved in a similar manner from a more ancestral monomeric unit of ca. 800 bp.