The northeast region of India, considered as ‘hot spot’ of biodiversity, having unique ecological environment with hot and high-humidity conditions, has given rise to the world’s hottest chilli, ‘Bhut Jolokia’, which is at least two times hotter than Red Savina Habanero in terms of Scoville heat units (SHU). This study was undertaken to determine the distinctiveness of ‘Bhut Jolokia’ from Capsicum frutescens or Capsicum chinense through sequencing of the ribosomal RNA (rRNA) gene-internal transcribed (ITS) region along with its phylogenetic analysis. Although a compensatory base change (CBC) in the ITS2 region was not observed between the closely related species of C. frutescens and C. chinense when compared with Bhut Jolokia; phylogenetic analysis using ITS1, 5.8S and ITS2 sequences indicated a distinct clade for all the accessions of ‘Bhut Joloikia’, while C. frutescens and C. chinense occupied discrete lineages. Further, a unique 13-base deletion was observed in all the representative accessions of ‘Bhut Jolokia’, making it distinct from all other members within the genus and beyond. The degree of genetic variations along with its extreme pungency might be related to ambient environmental factors of northeastern India.