The genus Isophya represents one of the largest orthopteran genera with about 45 species occurring in Europe. All over its range the genus includes groups of sibling species, the recognition of which is hampered by their morphological similarity. In part, some species were grouped according to their morphology: the Isophya straubei-group, the I. amplipennis-group and the I. major-group. Nevertheless,many species have not been studied using newmethods. This is particularly true for 15 taxa described from the Balkans, more or less resembling I. modesta, known mostly from old, unsatisfactorily informative descriptions. Chromosomal analysis of 25 species/subspecies of the genus Isophya showed the karyotypic evolution among species. The karyotype of 18 species/subspecies was investigated for the first time. The most remarkable changes in the sex chromosomes were the ancient acrocentric X chromosome inversions in 19 species and an X chromosome/autosome mutual tandem translocation, from which the sex determination system neo-XY originated in I. hemiptera. Karyotype differentiation has been less rapid in autosomes than in sex chromosomes, in this case interspecific autosomal differentiation has involved the distribution and quantity of C-heterochromatin as well as the number of NORs. Cytogenetic analysis revealed some changes reflecting the level of genomic organization,andtheresultssuggestsomeassociationofrelatedtaxafrombiogeographicregions.