OBJECTIVE The spatial distribution of cytotypes can provide valuable insights into evolutionary patterns of polyploid complexes. In a previous study the macro-scale distribution of the three main cytotypes in Senecio carniolicus (Asteraceae) within the Eastern Alps was characterized. Employing a roughly 12-fold extended sampling, the present study focuses on unravelling patterns of cytotype distribution on the meso- and microscale and on correlating those with ecological properties of the growing sites. METHODS DAPI flow cytometry of dried samples was used to determine DNA ploidy level in 5033 individuals from 100 populations spread over the entire Eastern Alpine distribution area of S. carniolicus. Descriptors of microhabitats as well as spatial data were recorded in the field, and analysed with a mixed-effects ANOVA. RESULTS Extensive variation in DNA ploidy levels (2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x) was detected. Of the main cytotypes, diploids and hexaploids were widespread and had strongly overlapping distributions resulting in the frequent occurrence of cytotype mixtures (half of the investigated populations), whereas tetraploids were disjunctly distributed and occurred in the south-west and the east of the species' distribution area. In spite of the frequent co-occurrence of cytotypes, only 1 % of the samples belonged to secondary cytotypes (3x, 5x, 7x, 8x, 9x). Diploids, tetraploids and hexaploids were altitudinally segregated, but with broad overlap. Similarly, highly significant differences in vegetation and rock cover as well as microhabitat exposure were found between the main cytotypes. CONCLUSIONS Senecio carniolicus shows a remarkable diversity of cytotypes. The distribution of the three main cytotypes (2x, 4x, 6x) has been shaped by Pleistocene glaciations to different extents. Whereas tetraploids are nearly entirely restricted to refugia, hexaploids colonized areas that were extensively glaciated. Diploid and hexaploid individuals often co-occur in mixed populations, where they are spatially and ecologically segregated at both the meso-scale (altitudinal differentiation, exposure of the growing site) and the micro-scale (cover of vegetation and bare rock). With regard to the ecological parameters investigated, the tetraploid cytotype occupies an intermediate position. The rareness of secondary cytotypes suggests the presence of strong pre- or post-zygotic mating barriers.