Aquilegia ecalcarata is the only spurless species within the genus Aquilegia and comprises a monophyletic clade with A. yabeana, A. kansuensis and A. rockii. Our previous study on the genetic diversity of those four species revealed that the populations of A. ecalcarata can be divided into two groups, indicating possible genetic difference within A. ecalcarata. However, it is not clear whether the genetic difference is related to the morphological variation among species and groups of A. ecalcarata populations. To answer that question, the morphological variation patterns based on 22 floral and 19 vegetative traits from 42 populations, covering the entire distribution of A. ecalcarata and its relatives were analysed in the present study. The result showed that (1) the differences among the four species were reflected in the floral rather than the vegetative traits; (2) populations of A. yabeana and A. rockii fell into one cluster each, and each of the six clusters occupied its own distribution range; (3) one of two A. ecalcarata clusters fell into a subgroup and shared common floral traits with A. rockii; (4) the individuals of A. ecalcarata form. ecalcarata and form. semicalcarata were often mixed in the same population; and (5) the populations of A. kansuensis were split into two clusters, which differed obviously in floral traits. These results will provide an important morphological basis for the redefinition of species and lay a foundation for the further exploration of the "spurless" A. ecalcarata.