The overwhelming effect of global warming is an alarming concern of food security and economic growth in Pakistan. Effective solution is of supreme importance to genetically analyse maize germplasm. Mainly maize relies heavily on traditional breeding methods that take several years before the release of suitable maize varieties in Pakistan. In order to improve the maize germplasm, knowledge of available gene pool and variability among them needs to be ascertained as a precursor for other developmental breeding programs to boost up crop yield. Present research was conducted at two temperature regime (Islamabad and D.I.Khan) i.e. relatively cooler and warmer zones that will serve as antecedent for further research to be conducted around the belt Islamabad to D.I.Khan. Screening on the basis of morphological and biochemical analyses were performed and further confirmed by utilization of SSR markers. Thirty out of sixty genotypes showed heat tolerance at both locations confirmed through PCR which can be a source material for breeding purpose to develop heat tolerant hybrid production to fulfil the potential yield gap.