The polyploid origin and colonization history of Iris aphylla in central and southeastern Europe were investigated by means of karyological and AFLP analyses. The study indicated two chromosome races within I. aphylla based on chromosomal numbers 2n = 24 and 2n = 48. The overall chromosome counts and determined ploidy suggest tetraploidy as the most common ploidy level for this species in Europe. Only one diploid I. aphylla population was found, with a restricted distribution area in the Slovak Karst. The analyses showed that tetraploids arose independently at least three or more times in the past. The karyological results were in agreement with the AFLP data and strongly suggested that migration between the two Carpathian Mts. sites occurred recently via the Dukielska Pass. The low number of AFLP fragments unique to each population supports the hypothesis of recent range expansion and colonization of different environments in central Europe by I. aphylla. All populations were genetically depauperated and had 0–3 unique bands, indicating that not enough time elapsed for many unique fragments to form through mutation. We also observed relatively high and significant differentiation between the one diploid and all investigated tetraploid I. aphylla populations (Φ_ST = 0.470, p < 0.001). The high and significant values of genetic differentiation can be explained by several factors such as anthropopression and natural succession, which have fragmented the open calcareous habitats in central Europe.