The sequence of centromere separation in late metaphase/anaphase cells from a long term culture of cells from a male rat-kangaroo, Potorus tridactylus, was studied. The karyotype of the PTK-1 line has five pairs of autosomes, one modified No. 1 (M1), and X, Y1 and Y2. In 87% of the cells the centromeres of No. 4 or 5 were among the first to separate. Y1, Y2, X, or the marker, M1, were not the earliest chromosomes to separate. Y2 separated last most frequently (59%), followed by X, M1 and Y1. The overall relative averages for the sequence of separation in the entire genome (centromere separation index or CSI) are 1.52, 1.40, 1.11, 0.92, 0.88, 0.86, 0.68, 0.56, and 0.27 for 5, 4, 2, 1, Y1, 3, M1, X, and Y2, respectively (the average for the whole genome is 1). The data indicate that the centromeres of the chromosomes of Potorus separate in a specific, genetically controlled, non-random sequence. The chromosomes separating late have larger amounts of centromeric heterochromatin; all of this or part of it may be the controlling element for the sequence of separation.