Tomato (Solanum lycopersicum L.) belongs to the family Solanaceae. It is consumed in differing ways, including crude, a fixing in numerous dishes, sauces and beverages. It thrives well in temperature 10 °C to 30 °C. The mean temperatures below 16 °C and above 35 °C are not desirable. In summer season temperature rises in most regions of province Punjab (Pakistan) up to 45 °C that affects the germination, pollination, fruit setting and development that ultimately lowers the yield and quality. There are different techniques used to select stress tolerant lines of tomato Viz. mutagenesis is one of a major technique to create variants of tomato which can be more stress tolerant. The present study was conducted on tomato cultivar "Rio Grande". Research experiments conducted were: 1) Development of mutant population through physical and chemical mutagens. 2) Screening of mutated M1 population for thermotolernace. 3) Raising of M2 population and its screening. 4) Assessment of genetic diversity/similarity among screened thermotolerant variants by using heat specific SSRs. EMS and Gamma irradiations were used to induce mutations. Germination percentage was observed in first experiment and concluded that only two doses of gamma irradiation 5 kr and 10 kr and EMS i.e. 4 mM, 8 mM, 16 mM, 24 mM and 32 mM showed germination and all remaining doses/concentration proved lethality in tomato seed germination. This mutated population was transferred into the field for development of M1 population and screening for thermotolerence was done by using different traits (membrane stability Index%, Cell viability, RWC, proline 2 contents, morphological, reproductive and fruit related traits). On the basis of all studied traits populations developed through gamma irradiation 5 kr and EMS 4 mM, 8 mM, 16 mM and 24 mM were selected to raise M2 population assessed for thermo tolerance by using same traits as in M1 population. On the basis of results obtained for thermo tolerance related traits, twenty four mutants were selected and raised as M3 population for assessing their genetic diversity/similarity with parent Rio Grande. On the basis of results obtained from all experiments, suitable doses and concentrations of physical and chemical mutagens were identified for mutation in tomato. Membrane stability index percentage and proline contents were found as suitable selection criteria for heat tolerance studies in tomato crop. Twenty four mutants were identified with enhanced thermotolerance which proved genetically distant from their parent “Rio Grande”.