Global population is increasing exponentially, however resources to support this population grow only arithmetically. The world’s population is expected to reach 9.7 billion by 2050. This projection increases the concerns related to feeding and managing the natural resources. In view of enhancing the productivity and fulfilling the basic nutritional demands of rising population, efficient and sustainable agricultural techniques must be employed. On a large scale, agricultural production is dependent on chemical fertilizers and pesticides to ameliorate the nutrient acquisition of plants and to control the pests and pathogens. However, these intensive agricultural techniques are environmentally hazardous, costly and unsustainable. Therefore, search for eco-friendly and sustainable alternatives are essential to meet the pursuit of global food security. One of the best alternatives include a unique group of fungi called endophytes which have revolutionized the world by their potent capabilities in diverse sectors, viz., agriculture, medicine, biotechnology and so on. However, the biggest problem lies with their identification as most of them do not sporulate in laboratory conditions and molecular methods alone sometimes misidentify them. In our study, besides novel combinations, a novel and effective technique (using cold treatment for the first time) was introduced to induce sporulation in endophytic fungi of a unique plant Ephedra gerardiana Wall. ex Stapf growing in extreme environmental conditions. The individual and synergistic effects of this technique (cold treatment) gave the best results in inducing sporulation. This will help in the correct identification of the isolates and accentuating better methods for endophytic fungal sporulation would not only solve the taxonomic position of these mycosymbionts but also lessen the time to make them sporulate along with unveiling their eco-physiological behaviour inside the host plant.