Karyotyping is the process of arranging pairs of the chromosomes in an order to examine chromosomal abnormalities which are the causes for Birth Defects. The overlapping and touching chromosomes of input metaphase spread images should be segmented and disentangled for this process. This paper proposes an algorithm to separate the touching chromosomes and the segmentation of overlapping chromosomes from G-Band metaspread images. Segmentation is performed by obtaining the interesting (concave) points on the image contour and constructing proper hypotheses for segmentation and separation.