Hordeum vulgare L. (barley) and Triticum aestivum L. (wheat) are among the most valuable crops cultivated and planted in many regions including Turkey. These plants have wide range of adaptation ability and capacity; they represent high level of variation in terms of physiological, genetics and epigenetics parameters and characteristics. Physiological and genetic variations were investigated by relative water content (RWC) assays, measuring electroconductivity (EC) levels and amplification of microsatellite markers in 21 barley and 43 wheat lines. At least three drought sensitive and three drought resistant lines were detected in barley and wheat lines via RWC assays. RWC values were recorded between 0.05±0.013 and 0.55±0.003%. Similarly great variation was detected for EC values of both barley and also wheat lines. Minimum and maximum EC values were ranged from 4.00±0.06 μS cm-1 to 59.88±3.209 μS cm-1. Three microsatellite markers, Bmag0120, Bmag0306 and Bmag375, were targeted in barley genome. Similarly, Han18, Wmc506 and Wmc623 microsatellite markers were targeted in wheat genome. Among these markers only Bmag0120 and Han18 were amplified from each line's genome by PCR and qPCR assays. In PCR and qPCR analysis homozygous and heterozygous lines were detected for Bmag0120 while each line was homozygous for Han18. Idiomorphic band size as 300 bp was detected in Han18 while it was ranged from 224 to 279 bp for Bmac0120 marker. Results showed that homozygous lines were drought resistant ones in barley lines whereas no correlation was found for wheat lines investigated in this study.