Isatis tinctoria L. was cultivated until the 19th century to produce indigo, a natural blue pigment used principally for dyestuffs. The current search for alternative crops and interest in natural products has led to reconsidering I. tinctoria as a crop to be grown in marginal areas to produce natural indigo. To reintroduce I. tinctoria into cultivation, its behaviour under different climatic conditions as well as its morpho-physiological and genetic diversity must be assessed in order to evaluate the possibilities of future breeding work. To do this, a Eurasian collection of 15 accessions was studied in a 2-year experiment. The study was carried out in four locations in order to assess plant performance at altitudes ranging from 380 to 1,700 m a.s.l. A second experiment evaluated the morpho-physiological diversity of several traits (some related to agronomic performances) of the collection. In a third experiment the genetic traits of the collection were characterised by using eight AFLP and eight SAMPL markers. The species showed a wide adaptability to different mountainous conditions and the populations showed high morphologic and genetic variability and differed according to their origins. Both morpho-physiological and molecular characterisation allowed the accessions to be distinguished into groups of European and Asian origin. Future breeding work is recommended because some accessions have good agronomic potential.