The core collection is a set of selected samples representing the genetic diversity in the base collection. This strategy has been proposed to minimize the conservation cost of genetic resources while preserving maximum genetic diversity. This study aims to determine the genetic diversity and formation of mini-core collection at Dryland Agricultural Research Institute (DARI) using morpho-physiological traits in lattice square design during 2014-2016. To this end, we surveyed 121 barley genotypes received from the National Gene Bank of Iran under both irrigated and rainfed conditions. Next, using factorial analysis, the studied 17 variables were distributed into 5 factors under both irrigated conditions and 4 extracted factors under rainfed conditions. The correlation analysis indicated a statistically significant positive correlation between morpho-physiological traits and grain yield under both conditions. Overall, five independent factors could explain 74% of the changes in irrigated conditions, whereas 5 independent factors together could explain 76% of changes under rainfed conditions. The mini-core collections consisting of 22 and 20 specimens were identified. For this purpose, important traits under rainfed and irrigated conditions with similar diversity to the initial collection were used, respectively. The diversity evaluation parameters in the core collection [mean difference percentage (MD%), coincidence rate percentage (CR%), variance difference percentage (VD%), and variable rate percentage (VR%)] in both mini-collections revealed that the diversity was well preserved in the initial collection in terms of generated mini-core collections. Therefore, it can be concluded that the mini-core collection selected from the initial germplasm can be very important for breeders to perform genetic designs. Accordingly, they can improve and introduce high-yielding cultivars and genotypes and important breeding traits of barley under rainfed (including drought) conditions.