61 sequences of cytochrome oxidase subunit 1 (COI), 570 bp long, from Central Europe (8), Slovenia (2), and from the Balkans (51: 6 from, Romania, 15 from Greece, 15 from Bulgaria, 6 from Serbia, and 9 from Montenegro), 33 of them new, together with 61 sequences of the ribosomal 18S, 450 bp long, all ofthem new, were analysed to infer the pattern of radiation of Bythinella in the Balkans. Thirty two nominal taxa of Bythinella (22 nominal species: B. austriaca, B. calimanica, B. charpentieri, B. compressa, B. dacica, B. dispersa, B.grossui, B. hansboetersi, B. luteola, B. micherdzinskii, B. molcsanyi, B. nonveilleri, B. pannonica, B. pesterica, B.radomani, B. rhodopensis, B. robiciana, B. schmidti, B. slaveyae, B. srednogorica, B. taraensis and B. viseuiana; one nominal subspecies B. austriaca ehrmanni; and nine Greek species not yet described) were included, represented mostly by paratypes or at least topotypes, collected at 31 Balkan localities. The phylogeny, inferred on the combined data set with the ML approach, showed two large clades, although they were weakly supported. One of them comprised the Romanian and Montenegro populations, and one Serbian population, the other (less genetically diversified) consisted of one Serbian and all the Bulgarian/Greek populations. The origin of those two clades was dated, with the external data, to be more than 4.341±0753 MYA old, thus its origin was assigned to isolation by the Dacic Basin (part of Paratethys). All the Bulgarian populations presumably belong to one species, which may be assigned to the recent recolonisation of this territory from the south.