Despite the ubiquitous occurrence of heat-shock protein 60 (Hsp60) and their role in maintenance of cell activity and integrity, this protein remains poorly characterized in many of the symbiotic soil mycorrhizal fungi such as Rhizophagus irregularis. Thus, in the current study, an attempt has been made to elucidate the evolutionary history, time of divergence followed by estimation of population genetic parameters of hsp60 using R. irregularis as a model organism. Sequence alignment reported here identified several close homologues for hsp60 (gene) and Hsp60 (protein) from diverse taxa, while the output from protein-based phylogenetic tree indicates that mitochondrial Hsp60 of R. irregularis shares close evolutionary relationship with classical α-proteobacteria. This is perhaps the first line of evidence elucidating the likelihood of hsp60 from fungal taxa sharing a close evolutionary relationship with classical α-proteobacteria as a common ancestor. Comprehensive analysis of mitochondrial hsp60 from selected fungal taxa from the evolutionary point of view explains the possibility of gene duplication and or horizontal gene transfer of this gene across various fungal species. Synteny relationships and population genetics credibly explain high genetic variability associated with fungal hsp60 presumably brought by random genetic recombination events. The results presented here also confirm a high level of genetic differentiation of hsp60 among all the three fungal populations analysed. In this context, the outcome of the current study, basedon computational approach, stands as a testimony for explaining the possibility of increased genetic differentiation experienced by hsp60 of R. irregularis.