Drought is a major agronomic problem requiring immediate efforts to solve, to detect its effects on crop productivity. Tissue culture and mutagenic potentiality of EMS (ethyl methane sulfonate) were used to develop rice-promising drought-tolerant lines. Mature embryos of two genotypes (Sakha 101 and Giza 177) of rice were used for developing callus. Three callus induction strategies were investigated by applying EMS as a mutagen to evaluate and determine the best procedure for selection of drought-tolerant rice cell lines. For screening of calli response to PEG (polyethylene glycol), two-months-old well-proliferated treated calli of three protocols were sub-cultured for two weeks with 5% and 7% PEG-6000. Molecular response to PEG was evaluated by detecting gene expression of rice dehydrin, phytoglobin 1, 2, and 5 genes via qRT-PCR (quantitative reverse transcription-polymerase chain reaction). The relative gene expression of all genes was significantly increased in the PEG-treated calli compared with the control in both genotypes and Giza 177 showed higher expression levels than Sakha 101. Genetic variations were assessed among three putative mutants arising in vitro, their mother plants, and a drought-tolerant genotype (IET 1444) using ISSR fingerprinting. Similarity coefficients reflected the genetic relationship between rice regenerants and their mother plants. Cluster analysis showed regenerated Sakha 101 mutant line and IET 1444 were grouped together at a dissimilarity distance of 1.00. In vitro screening of EMS mutants with the creation of chemical drought using PEG-6000 to assess tolerance could be a good track to developing drought-tolerant rice lines.