Viola tricolor is a pseudometallophyte covering heavy-metal-polluted and non-polluted areas. The species is a member of the evolutionarily young sect. Melanium of Viola. In this study, we sought to determine whether the karyotype of V. tricolor is stable with respect to chromosome structure or is altered depending on environmental conditions (heavy-metal-polluted vs. non-polluted areas). We established the karyotypes of plant material originating from a Zakopane meadow (non-metallicolous population) and from the Bukowno mine waste heap (metallicolous population), showing evident interpopulation differentiation in chromosome type (2M+20 m+2sm+2st vs. 18 m+8sm), in the number, size, and distribution of rDNA loci (25S and 5S), and also in chromosome mutations, mainly fission of chromosomes into acentric fragments and translocation of the fragments. Variable numbers of both 25S and 5S rDNA loci were distributed at different positions of the chromosomes and not on specific pairs of chromosomes. The results clearly indicate that the karyotype of V. tricolor results from the unstable genetic structure of the species. This character, typical for relatively young evolutionary groups, proves its membership to the Melanium section considered to be young within the genus Viola.