Milk Thistle ( Silybum marianum ) is a drought tolerant medicinal plant which has a high distribution in Iran. Little is known about genetic diversity and identification of drought tolerant ecotypes in Iran. For this purpose, 25 ecotypes of milk thistle from eight provinces of country and one ecotype from Hungary were evaluated in two different moisture conditions (non-stress and drought stress during 2013-2014. Results showed that there was high variability among ecotypes for most of the studied traits. The highest coefficient of variation and broad sense heritability in both moisture conditions was related to trait of capitulum number in plant. Average of most of the traits in stress levels compared to non-stress conditions showed reduction, but the average traits of the main capitulum diameter and biochemical traits (including oil and silymarin content) increased under drought stress condition. Grain yield had 34 percent reduction due to drought stress. Under normal moisture conditions, ecotypes 15 and 24 from Khuzestan province and under drought stress level, ecotype 18 from Khuzestan province showed the highest average grain yield. Under none-stress moisture conditions, the correlation between grain yield and capitulum number, oil percentage and silymarin yield was positively sgnificant. Under drought stress condition, grain yield was positively correlated with plant height, capitulum number and silymarin yield. In general, phenological traits had negative and significant correlation with the biochemical traits. The results of stepwise regression showed that under non-stress condition the capitulum number per plant and main capitulum diameter and under drought stress condition the capitulum number perplant and number of seed per capitulum justified the most variation of grain yield. Generally, in both moisture conditions capitulum number had more effect on grain yield and can be used for indirect selection in breeding programs. The result of cluster analysis, based on morphological and biochemical traits in both moisture levels could mainly separate ecotypes based on geographical origins. Considering the positive and significant correlation of STI, GMP and MP indices with grain yield in normal and stress conditions, these indices were identified as suitable criteria for selection of drought tolerant genotypes. The results of mean comparison of tolerance and susceptibility indices showed that based on STI index, ecotype 18 from Khuzestan was the most tolerant ecotype and ecotype 9 from Guilan was identified as the most sensitive ecotype to drought stress. Keywords : Milk thistle, genetic diversity, drought stress, oil, Silymarin content