Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and inter simple sequence repeats (ISSRs) markers were performed to detect the genetic diversity among 6 new rice lines and 4 cultivars with different responses to drought tolerance and establish specific DNA markers associated with drought tolerance. Among 16 RAPD primers tested, only 5 produced bands polymorphic between lines with an average of 5.2 bands per primer (ranging from approximately 252 to 1232 bp) and 73.02 % were polymorphic. Among the tested ISSR primers, only five amplified polymorphic ISSR loci with an average number of 4.4 bands per primer (ranging from approximately 80 to 813 bp) and the mean percentage of ISSR polymorphism was 90.91. Based on band polymorphisms generated by RAPD-PCR and ISSR after using the primers, the highest similarity value (0.93) was found between P-5-3-b line and P-5-3-a line and the lowest value (0.44) was found between P-5-3-b line and Giza 172. The dendrogram separated all cultivars and new lines into two clusters and indicated that the cross of tolerant line (P-5-3-b) and susceptible cultivar (Giza 172) is suggested as the most suitable cross for drought tolerance analysis studies as they have the lowest similarity value (0.44) and also grouped in distinct cluster. Since two fragments of about (315 and 505 bp) were visualized using HP15 primer in the genomic DNA of the drought tolerant lines while were absent in the sensitive cultivars, they can be considered as positive drought tolerant markers. [Journal of American Science 2010; 6(12): 355-363]. (ISSN: 1545-1003).