Background: Mothbean is minor pulse crop grown in arid and semi-arid region. Mothbean wilt is one of the most important diseases directly contributing to the yield losses of the crop. It is caused by Fusarium which shows the great cultural, morphological and molecular diversity. Nowadays, the meagre information known mothbean wilt and pathogen causing it. Methods: Present investigation was conducted during 2022-2023, the fixed plot survey was carried out in moth bean growing farmers’ fields of Bharuch district. Eleven isolates of the pathogen were collected from selected villages of Bharuch district. The cultural and morphological characteristics of pathogen were studied under laboratory condition as per standard methods. The molecular variability of eleven isolates causing mothbean wilt was studied employing standard six ISSR primers. Result: During this investigation, the pathogen causing mothbean wilt is new record from South Gujarat location. Eleven Fusarium isolates causing mothbean wilt was found under Bharuch district of Gujarat. The cultural, morphological and molecular characteristics of eleven isolates were studied during present work. The present investigation will explore detailed study of Fusarium isolates causing mothbean wilt and their geographical cultural and morphological characteristics along with molecular diversity.