The genus Pseudophoxinus Bleeker, 1860 is found in a wide range of habitats in central Anatolia, but it is not well known from a cytogenetic aspect. In this study the first karyotypic description of the spring minnows Pseudophoxinus crassus (Ladiges, 1960) and P. hittitorum Freyhof & Ozulug, 2010 by means of conventional methods (Giemsa staining, C-banding, silver nitrate impregnation (Ag-NORs)) was performed. Both species are endemic and have restricted distributions in Central Anatolia. P. crassus and P. hittitorum have the same diploid chromosome number, 2n = 50, patterns of distribution of constitutive heterochromatin (CH), and localization of nucleolus organizer regions (NORs), but differ in their karyotypic formulae (KFs). The C-banding technique revealed clear pericentromeric blocks of CH in many chromosomes; Ag-NORs treatment revealed consistent positive signals at the end of the short arms of a submetacentric chromosome pair, likely homologous in both species. The karyotypic differences found between these species can be used for their taxonomical study.