Blastocystis sp. is the most common enteric protist colonizing a wide range of host and is characterized by wide genotypic diversity. Heat Shock Protein 70 (HSP70), a common stress-response protein has been associated with increased proliferation in Blastocystis sp. under thermal stress. However, the extent of HSP70 genetic variation and its link to thermostability remains unexplored. Our study aims to elucidate HSP70 gene variants among different Blastocystis sp. subtypes, analyze their responses to thermal stress, and evaluate their protease activity following the thermal stress. Heat Shock Protein 70 gene from eighteen Blastocystis sp. isolates of varying subtype – ST1, ST3, ST4, ST6, and ST7 – were amplified, sequenced and phylogenetically analyzed. The cells were then subjected to thermal stress at 42°C for 24 hours and the cell viability was monitored for seven days. Protease activity in solubilized antigens (SA) from purified Blastocystis sp. cells was evaluated following exposure to thermal stress. Phylogenetic analysis revealed two distinct clades: ST4 and ST7 (Clade 1) which are mostly thermotolerant, while ST1, ST3, and ST6 (Clade 2) exhibited lower tolerance. Total protease activity was similar across subtypes, suggesting need for further sub-class specific analysis. Our findings suggests that thermal stress adaptation is subtype dependent and may be influenced by HSP70 variation. This insight highlights the importance of HSP70 as a potential molecular marker for stress adaptation in Blastocystis sp. and underscore the need for deeper investigation of its functional role in parasite persistence and pathogenic potential.