Wilt disease, affecting mango trees (Mangifera indica L.), is recognized as sudden death of mango. This epidemic was reported first time in Punjab (Muzafargarh), Pakistan in 1995. High disease severity is recorded in nearly all mango producing areas of Punjab and Sindh ranging from 2-5% and 5-10%, respectively. Though the incidence of the disease has been significantly lowered due to the mass awareness and development project and programs of Mango sudden death (MSD) disease throughout the country. Work on the pathogen causing MSD is essentially concluded but the relationship of Ceratocystis with MSD and the species involved is still not well studied in Pakistan. The present study focused on geographical distribution, pathological and genetic characterization of Ceratocystis sp. associated with MSD, for this a survey was done for the collection of samples during the year 2011-12. From different mango growing areas of Punjab and Sindh forty-five Ceratocystis species isolates were obtained. Pathogenicity of these isolates were done on potato tubers, fresh mango leaves (detached), fresh green mango twigs (detaced) and white chunsa mango seedlings. All isolates incited symptoms with varying amount of virulence in contrast to control. More prominent lesions were witnessed on potato tubers (0.439-7.53 cm2) followed by mango seedlings (0.84-4.39 cm2). Deoxyribose nucleic acid (DNA) from all 45 isolates was effectively extracted by using phenol chloroform method. Comparison of DNA sequence using internal transcribed spacer (ITS), beta tubulin (BT) gene, and Transcription Elongation Factor (TEF) gene primers confirmed that Pakistan isolates were dissimilar from Ceratocystis fimbriata and Ceratocystis omanensis reported previously. The fungus made one of intensely supported sub-clades through phylogenetic tree. On the basis of morphological characteristics and assessment of DNA sequences for three gene regions, it was concluded that the Ceratocystis species related with MSD in Pakistan is Ceratocystis manginecans.