<p>Poultry farming in high altitude like Leh-Ladakh, India is a challenge due to hypobaric–hypoxia environment and probiotic may support gut health, feed efficiency, production and adaptation in chicken. Therefore, the present study was performed to elucidate the role of probiotic on broiler chicken gut microflora from two different high altitude locations; Leh and Chang La. No change was found on the growth of the broiler with probiotic intervention. The fecal microbiota study revealed that the microbial abundance was higher in Chang La samples as compared to Leh samples. At taxonomic level, Chang La samples were dominated by <i>Firmicutes, Proteobacteria, Bacteroides, Actinobacteria</i> phylum whereas in Leh samples, <i>Elusimicrobia</i> was dominant followed by <i>Nitrospirae</i>. Probiotic treatment had minimal effect on chicken gut microflora at the genus and species level. The probiotic effect on gut microflora of Leh broilers was more diverse as compared to the control group. Contrary, to the Chang La group, the probiotic effect had less diverse microbial population as compared to the control group; maintaining the gut microbiome homeostasis of broiler chicken in such extreme high altitude. The microbial functional annotation revealed differences majorly in pathways related to maintenance of cellular energy homeostasis, glycan and carbohydrate metabolisms, regulatory systems, signaling pathways, environmental processing and cell–cell signaling. The findings of the study are an indicative of diversified gut microflora of broiler chicken from two distinct high altitude locations. The study can further be useful to explore the role of different probiotic strains, their doses or delivery methods for the adaptation of these birds at such extreme environments.</p>