DNA analysis of specific regions in chromosome is important for acquirement of valuable genes and study of genetic diseases. Recently, atomic force microscopy (AFM) has also been applied to the chromosome dissection and the recovery of the chromosome fragments. However, any recovery method with high reproducibility by AFM has not been established yet. We developed a highly reproducible dissection and recovery method for chromosome fragments by using AFM, named “scratch method”. This method was available for different sized chromosomes both silkworm chromosome in pachytene phase and human metaphase chromosome of somatic cell. The scratch method will contribute to a development of more efficient and rapid DNA analysis in future.