The aim of present study was to investigate the genetic variation within and between 32 milk thistle ecotypes collected from northern (23 accessions) and southern (9 accessions) regions of Iran along with two introduced varieties, CN seeds and Budakalaszi, for morphological and flavonolignans properties. The two collections were assessed at separate field experiments. MANOVA for all the morphological traits showed significant difference between ecotypes. Univariate ANOVA verified these differences for most of the traits in the northern ecotypes (first collection) while for southern ecotypes no significant differences were obtained for the studied traits except seed yield. Among and within ecotypes genotypic coefficient of variation indicated higher level of variation among ecotypes than within ecotypes. In both of the experiments, there was a large genetic variation for silybin and silymarin quality and quantity. Cluster analysis of 34 accessions was performed for morphological traits and silymarin and silybin characteristics, separately. The resulting dendrogram based on silybin and silymarin characteristics revealed that the native accessions such as Dezfoul, Fereydounkenar and Nour, had highest flavonolignans and they were better than the foreign varieties. Also, there was no clear relationship between clustering based on morphological traits and flavonolignan compounds.