Aims -Iran is one of the main centres of diversity for the genus Onobrychis Miller. This study includes 55 specimens from eleven representative wild populations of Onobrychis carduchorum C.C.Towns. originating from their natural habitats in Iran. The aims of this study are to provide a detailed taxonomical, morphological, genetic and ecogeographical characterization of O. carduchorum in Iran. Methods -The specimens which represent all Iranian populations were biometrically assessed using 45 quantitative and fifteen qualitative morphological characters. The phenotypic variation among the populations depends on various environmental factors; thus at each sample site we recorded data regarding longitude, latitude, altitude, slope inclination, slope orientation, substrate, minimum and maximum annual temperatures, number of rainy days, annual precipitation as well as soil traits including texture, electrical conductivity, organic carbon, total nitrogen, available phosphorus, available potassium, total neutralizing value, pH, and saturation percentage. The floristic composition of each population habitat was examined as an indicator of environmental conditions. To assess genetic variation, we obtained nucleotide sequence data from the internal transcribed spacer of the nuclear ribosomal DNA (ITS) and carried out genomic fingerprints using inter-simple sequence repeat (ISSR) analysis. Results -Cluster analysis of morphological characters showed that the eleven populations could be divided into two major groups including five subgroups. Principal component analysis (PCA) of floristic data confirmed the two major morphological groups suggesting habitudinal segregation among the groups and the indicative value of floristic composition of study sites in assessing intraspecific variation in the target species. Furthermore, canonical correspondence analysis (CCA) of ecogeographic data showed correlations between morphological variations and ecogeographic factors. Longitude, latitude, substrate, available potassium, clay%, total nitrogen, organic carbon, slope orientation, sand%, texture, altitude and rainy days are apparently the main environmental variables associated with morphological groups of O. carduchorum. Both ITS and ISSR data indicate that O. carduchorum is a young species with a recent divergence of its populations.