The mitotic effect of physical mutagen gamma rays was observed in the root tip cells of Ashwagandha. The Chromosome analysis has been showed as an important tool for establish variability of the plant seed by use of physical mutagen gamma rays. The gamma rays have of low wavelength and high penetrable power. The plant has tremendous medicinal values and it is known from ancient times. The dry and well matured seeds of ashwagandha were irradiated with different doses of gamma rays viz., 5,10,15,20,25,30,35, 40, 45 and 50 KR respectively. The chromosome number of control plant is 2n = 48. The gamma rays affect the normal cytological behavior of ashwagandha species. The chromosomal aberrations increase with increase in the doses of gamma rays to optimum level of 30KR, because it causes changes in the chromosome structure, cellular structure and metabolism of plants. The chromosome aberration like, Sticky metaphase, Precocious moment chromosome, Fragments, Anaphasic bridge, Anaphasic laggard, Telophasic laggard. The present investigation was carried out to study the cytogenetic analysis of the species Withania somnifera. The chromosomal aberration increases with increase in the doses to optimum level (50 KR) of physical mutagen gamma rays.