For the first time, a conventional analysis of C-banded karyotypes was carried out in two distantly related diplopod species; this revealed an impressive percentage of heterochromatin in both genomes. In Acanthopetalum sicanum (Order Callipodida) (2n = 12), heterochromatin constitutes about 60% of the total DNA in females and 56% in males, whereas in Enologus oxypygum (Order Julida) (2n = 22) it is about 67% in both sexes. Heterochromatin of the two species was found to be similar in base composition (AT rich) and heterochromatin distribution, indicating that it has accumulated in a species-specific manner. Sex-determining mechanisms of the XY type were detected in both A. sicanum and E. oxypygum. In A. sicanum, the Y presented the lowest heterochromatic content of all chromosomes in the karyotype, whereas the X presented the highest.