A hypothesis on the selective neutrality of relative lengths of karyotype chromosomes was tested. Idiograms expected based on an assumption of selective neutrality of chromosome lengths were compared with actual idiograms in more than a hundred mammalian species. The observed idiograms differed from those expected in a similar manner: in the observed idiograms, the longest chromosomes were shorter, and the shortest were longer than expected. It is suggested that karyotype chromosome variation is limited by selection against chromosome rearrangements that produce very long or very short chromosomes. An analysis of reciprocal translocations in the mouse and Drosophila showed that translocations generating chromosomes of extreme lengths were more deleterious than those generating normal-sized chromosomes. A working hypothesis was advanced stating that within-karyotype variation of chromosome lengths is accounted for by two factors: chromosome rearrangements and natural selection. Chromosome rearrangements tend to randomize relative chromosome lengths in a karyotype, whereas natural selection acts to equalize them.