Polyploidization is generally considered to be an important evolutionary driver affecting the genetic diversity, that can alter the morphology, phenology, physiology or ecology of plants, which in turn may make the taxonomy of polyploids more difficult. One such example is the Symphytum officinale complex, a polyploid species group represented by three major cytotypes: tetraploids (2n = 48), less common, geographically restricted diploids (2n = 24) and hypotetraploids (2n = 40). In most European floras only one polymorphic species, S. officinale, is widely recognised, while the particular cytotypes are usually considered conspecific. Our study provided a thorough evaluation of the ploidy level diversity, morphological and ecological variation, with a special attempt to clarify the status of “white-flowered” diploids. Using flow cytometry, we identified three cytotypes: widespread tetraploids (76.1%); less frequent diploids (23.6%) with scattered distribution across the range of tetraploids and confined only to several areas of Europe; and extremely rare triploids (0.3%). Diploids and tetraploids showed diffuse parapatric pattern of distribution, with only four mixed-cytotype populations (2.7%) found, but almost entirely without triploids, suggesting reproductive isolation between di- and tetraploids. Niche of diploids falls nearly completely within the niche of tetraploids that showed niche expansion. Tetraploids also showed a shift in niche optimum toward a less continental and colder climate, coupled with expansion to more disturbance-prone sites with higher nutrient availability. Diploids were clearly distinguishable morphologically from tetraploids. The morphological differentiation of studied cytotypes appears to be taxonomically significant, especially in combination with ecological segregation and the apparent presence of hybridisation barriers. Both cytotypes should be treated as separate species (i.e. S. bohemicum F.W. Schmidt and S. officinale s. str.).