Cereal rust is one of the most damaging diseases of small-grain cereals. The fungus Puccinia hordei causes leaf rust in barley and other small grain crops. Puccinia hordei causes serious yield losses in the cultivating areas where susceptible and late-maturing barley varieties are cultivated. Therefore, rust-resistant barley cultivar is highly demandable for sustainable small-grain crop production. Improving barley yields and quality is one of the major objectives of barley breeding programs in our country. Exotic and indigenous germplasm is one of the best sources of resistance to biotic stresses in barley particularly leaf rust caused by Puccinia hordei. Hence, the present investigation was carried out to identify the resistance sources to P. hordei and incorporate them into the breeding programs for higher barley yields under changing climatic scenarios. The study aimed to identify new resistant cultivars in barley and other small grain crops. In this study, 100 barley genotypes (Hordeum vulgare L.) were considered for screening susceptibility to P. hordei causing rust disease. Several biochemical responses were analyzed in P. hordei infected barley genotypes. However, the variable response was observed among the 100 barley genotypes while those were screened against leaf rust disease under high altitude cold arid conditions of Ladakh, India. The efficiency of the 100 barley genotypes were categorized into different classes including high resistance (4 genotypes)>resistance (14 genotypes)> moderately resistance (20 genotypes)> moderately susceptible (33 genotypes)>moderately susceptible to susceptible (19 genotypes)> and susceptible (10 genotypes) based on plant response to P. hordei. Among the total genotypes, SHEIKH/KP-706, SHEIKH-B1, SHEIKH-636, and IC-062190 showed high resistance (8.07-8.63) as per the international leaf rust scale, while EC-667381, EC-667390, EC-667392, EC667396, EC-667417, Jyoti, EC-667434, EC-667442, EC-667445, and EC-667446 were found as susceptible (3.13-3.97) to P. hordei. The highly resistant genotypes accumulated a high level of phenols and flavonoids and cooperated with susceptible and other rest of the genotypes in response to P. hordei rust. The efficiency of plant immune response and or fitness to P. hordei was correlated to the disease susceptibility index of particular genotypes. This provides a new insight and the mechanistic basis of genotype-specific rust disease susceptibility against P. hordei. A large number of genotype-based studies at the field level could be useful to plant breeders and farmers for improving rust resistance in barley and other small-grain cereals.