Chili (Capsicum annuum L.) is the most widely grown solanaceous crop in the world. However, their production has reduced over several years due to the attack of various fungal and bacterial pathogens and various abiotic factors. Still, the major constraints in their production are pathogens with fungal etiology, especially the fungal wilt of solanaceous crops. One among the fungal diseases is Fusarium wilt, caused by the Fusarium oxysporum has emerged as a serious problem in past decade (Anonymous, 2005). (Singh, 1998) first time reported the wilt disease of chili caused by Fusarium spp. Fusarium oxysporum, F. solani, F. moniliforme and F. pallidoroseum have been reported as the wilt causing agents from chili growing areas but in India F. oxysporum and F. solani are the most prevalent species of Fusarium found associated with wilt disease of chili (Madhukar, 2004). Fusarium oxysporum have been previously identified as the pathogens causing wilt disease in chili. Recently, a new fungal pathogen F. equiseti has been reported as the causal agent of wilt disease infecting chili. DNA extraction, PCR amplification, and sequencing were performed on the various diseased plants to isolate the fungus. DNA barcoding using the internal transcribed spacer region (ITS) was used to identify the pathogen followed by the pathogenicity test. Further confirmation of the pathogen was done by sequencing of transcription elongation factor (TEF) and Calmodulin (CAL2).