Alyssum ovirense (Brassicaceae) is disjunctly distributed in the eastern Alps, predominantly occurring in the southern limestone Alps, but with one isolated population on the Hochschwab massif in the northern limestone Alps. The closely related rare and narrow endemic A. wulfenianum is restricted to gravel beds in a few rivers in the southern Alps draining the distribution area of A. ovirense. Applying molecular (amplified fragment length polymorphism fingerprinting and sequencing of plastid and nuclear DNA), karyological (flow cytometry and chromosome counts) and morphometric methods to a set of populations sampled throughout the distribution range, we evaluated the status of the population on the Hochschwab massif and the relationship between high-elevation A. ovirense and low-elevation A. wulfenianum. The population on Hochschwab massif is hexaploid, morphologically clearly divergent and grows in vegetation patches and dense alpine grasslands, whereas southern Alpine A. ovirense inhabits sparsely vegetated limestone screes. Molecular data show that the former represents a genetically isolated lineage which is formally described as A. neglectum sp. nov. All populations from the southern limestone Alps, including A. wulfenianum, are diploid. Although A. wulfenianum is genetically nested in A. ovirense, its morphological and ecological differentiation justifies recognition at the subspecific level. As A. wulfenianum has priority over A. ovirense, the correct names are A. wulfenianum subsp. wulfenianum and A. wulfenianum subsp. ovirense. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 486–505.