Presence of polysaccharides and polyphenolic compounds in Aconitum balfourii proved as hindrance in genomic DNA isolation. Different protocols were tried but DNA isolated was contaminated and pigmented. The present research work deals with optimization of DNA isolation and genetic analysis of Aconitum balfourii Stapf. on high yields of pure DNA samples. Different protocols were modified in such a way that polyphenolic compounds are removed before precipitation of DNA with isopropanol. DNA obtained is of good quality and is ready to use for PCR amplifications, RAPD and ISSR. As protocol is simple inexpensive and reproducible so can be used routinely.