C-banding is a method used for studying chromosome rearrangements near centromeres and for investigating polymorphisms. In human chromosomes, the C-bands are located at the centromere of all the chromosomes and the distal long arm of the Y chromosome. In this study, we aimed to detect the structural changes in chromosomes during the stages of C-banding by atomic force microscopy. We observed crater-like structures in the chromosomes after 2xSSC (saline sodium citrate) treatment and measured the relative difference between the heights of chromatid and centromere of the chromosomes. Results showed that the relative difference was 3 nm in chromosomes 1, 9, 16, and Y, whereas in the other chromosomes this value was 11.6 nm. After Giemsa staining, the relative difference increased by a factor of 16 in chromosomes 1,9, 16,and Y. The other chromosomes showed no such increase,which is in accordance with our suggestion that nonhiston proteins associated with DNA in constitutive heterochromatin can make the constitutive heterochromatin resistant to C-banding.