Pomegranate (Punica granatum L.) is an important fruit crop farmed all over the world, notably in the tropical and subtropical zones. It is afflicted by a number of diseases, the most serious of which is Ceratocystis fimbriata. Very little work is done on the genetical variability of C. fimbriata associated with pomegranate wilt in Karnataka and the morphological structures defining this species are reasonably defined, these characteristics are insufficient to recognize emergent grouping within C. fimbriata. The aim of this study is to confirm the identity of C. fimbriata isolates from pomegranate in Karnataka and to compare the DNA sequence of the internal transcribed spacer (ITS-1 and 4 regions) variability is the property of an organism to change its characters from one generation to the other. there is a need to study C. fimbriata based on molecular variability. Fifty isolates of C. fimbriata were amplified with a range of 600-650 bp length, twelve isolates were sequenced and deposited in the GenBank Maryland, USA database under the accession number KY038512- KY038523. All isolates from different districts of Karnataka show specific patterns of similarity according to geographical region.