Despite the substantial knowledge of the variation in cytotypes at large spatial scales for many plants, little is known about the rates at which novel cytotypes arise or the frequencies and distributions of cytotypes at local spatial scales. The frequency distribution, local spatial structure, and role of habitat differentiation of tetra-, penta- and hexaploid cytotypes of the bulbous geophyte Allium oleraceum were assessed in 21 populations sampled in the Czech Republic. The ploidy levels determined by flow cytometry confirmed that there was a mixture consisting of two or three cytotypes (i.e. 4x+5x, 4x+6x, 5x+6x, 4x+5x+6x). In addition, mixtures of cytotypes were found at sites previously considered to be cytotype-homogeneous. At all sites previously found to contain a mixture of two cytotypes, no plants with the third ploidy level were found. Although the relative frequencies of cytotypes varied considerably both among and within populations, mixed populations consisting of tetra- and hexaploids were usually dominated by tetraploids. This suggests that there are secondary contacts among cytotypes but there is little gene flow among them except for the rare formation of hexaploids in tetraploid populations. Cytotypes were not randomly distributed over the study area but were spatially segregated at either 47.6% or 61.9% of the sites investigated, depending on the statistical test (Mantel test or average distance test) used. When the composition of habitats at each of the sites is taken into account, cytotypes were more frequently spatially segregated at sites with a heterogeneous environment than a homogeneous environment. This implies that the cytotypes are ecologically differentiated. The frequent co-occurrence of cytotypes, with or without significant spatial segregation, at many sites with heterogeneous or homogeneous environments, however, suggests that niche differentiation alone is probably ineffective in determining co-occurrence. It is supposed that the prevailing vegetative reproduction associated with local dispersal, a high population density of the species in a landscape, and non-equilibrial processes influencing the establishment and extinction of A. oleraceum populations can also support the local co-occurrence of cytotypes.