Despite its complicated taxonomy, the genus Hesperis has not yet been subjected to any detailed molecular phylogenetic study and little is known about its monophyly, origin and biogeographical history, as well as the evolution of morphological characters. Here, we present for the first time comprehensive molecular analyses (nuclear ribosomal ITS sequences) of approximately 40 Hesperis species which represent the full range of morphological variation and the entire geographic distribution area of the genus. Based on our results, monophyly of the genus Hesperis has been proved. Moreover, our phylogenetic analysis shows that almost all traditionally defined sections are not monophyletic. NeighborNet network analysis was performed in order to visualize conflicting phylogenetic signals. The split-graph corresponds to the major clades represented in the consensus tree and revealed a hybridization signal in the evolutionary history of only a single Hesperis species. Divergence time estimations indicate that the origin of Hesperis (7.66–19.9 Mya) coincides with the expansion of grasslands and the closure of the proto-Mediterranean Sea in the middle Miocene. Therefore, diversification within Hesperis was affected by global climate changes, substantial tectonic rearrangements and the expansion of open vegetation systems in the Miocene, all of which had a great impact on the speciation history of the Irano-Turanian flora and fauna. Furthermore, two new species from West of Iran are described and illustrated here. They are readily distinguished from their closely related species by morphological characters such as stem height, leaf shape, pedicel, sepal and fruit indumentum, petal size, stigma shape and seed characters.