B arely is an important cereal crop and ranks around fourth in the world cereal production statistics following wheat, maize and rice (USDA-2008). It is estimated that annually over 133.85 million tones barley is produced worldwide. Barley is used as feed, food and for the brewing and distilling industries (Morris and Bryce, 2000). Barley is containing 80% carbohydrates which is an important source of energy. In the roman times, barley was the most popular food of providing a high energy, before going to the circus. The gladiators were fed on barley diet, so they were called "hordeari" from hordeium. Researchers also found that the amount of cholesterol in humans is decreased in blood serum due to the consistency of Abstract | Genetic diversity can be investigated through different methods such as morphological traits, biochemical (SDS PAGE) and molecular markers. The present work was conducted on the basis of morphological and biochemical characterization in order to estimate the genetic diversity among the barley landraces; to explore significant variation which can be used in breeding programs. For this purpose, 40 barley landraces were collected from Dir Lower and Swat, districts of KP, Pakistan. A total of 18 traits were recorded, 10 qualitative characters including spike density, awn barbs, glume awn, lemma type and awn color, length of the rechilla hairs, lemma color, auricle pigmentation, photoperiod sensitivity and stem pigmentation. Variation was observed in these qualitative traits through frequency distribution. In quantitative traits maximum variation was found for plant height ranging from 36cm to 50cm, days to flowering range from 130 to 133 days), while 100 seed weight ranged from 36 to 86 g and plant biomass range from 50 to 199.5 g per plant. SDS-PAGE analyses of total seed storage protein resulted in a total of 18 polymorphic bands. Total genetic diversity on the basis of total seed storage protein analysis was 17.5%. In Band, the 14 total genetic diversity was (0.60%) followed by Band 16 (0.58%) and Band 17 (0.55%). Similarly, Band 3 showed diversity, while Band 8, 4 and 5 indicated 0.45, 0.38 and 0.35%, respectively. A cluster dendrogram tree was constructed which was divided into two linkage and further divided into seven clusters. The variation was found among the clusters. The information obtained from the study is useful in planning further crop improvement.