Geographical variation of cytochrome b mitochondrial DNA (mtDNA) in chub (Leuciscus cephalus L.) was analysed in 31 samples from 20 different river basins in the Balkan Peninsula and Danube catchment. Multivariate methods of ordination were used to analyse variation of
the data sets. The results were interpreted in the context of the proposed ichthyogcographic districts separating the Balkan Peninsula into two main ichthyogeographic divisions (Eastern Greece/Ponto‐Acgcan and Western Greece/South Adriatic‐Ionian). Boundary detection supported
these two ichthyogeographic districts for L. cephalus, revealing a boundary that ran from north to south through the Balkan Peninsula and the middle of Greece. The results also revealed the existence of a third division in Central Greece. The results of ordination techniques on homogeneous
zones and analysis of the molecular variance confirmed the results obtained in studying local variability (boundaries). They also allowed us to test the existence of possible subdivisions proposed by different authors inside the two main ichthyogeographic districts. These subdivisions were
not supported. The multivariate methods used in this study allowed us to propose a coherent picture of chub ichthyogeographic districts in terms of boundary detection and maximal autocorrelation between populations and to explain the patterns of chub mtDNA variation. A complete interpretation
of results concerning L. cephalus requires careful consideration of both boundary analysis and autocorrelative approach. Results from an the autocorrclativc approach alone could lead to substantial misinterpretations.