Karyotyping helps to evaluate the size, shape and number of chromosomes. It is a screening and diagnostic process for finding various abnormalities related with chromosomes. Banding pattern is unique for each pair of chromosome. In the present research, fractional derivatives find an important place in the field of signal processing and digital image processing. This paper proposes a method for segmenting the dark bands in chromosome using fractional order derivative. First, enhancement of image is done to improve the visual quality of dark bands in chromosomes. Then fractional order derivative is applied to the enhanced image for segmenting the dark bands in chromosome. The experimental results clearly exploit thatFractional order differential mask operators achievean increase in PSNR and improvement in terms of visual quality of the images compared to integer order derivatives.