The cytogenetic characteristics of the grasshopper Podisma sapporensis (two races 2n = 23♂ X0/XX and 2n = 22♂ neo-XY/neo-XX) were analysed through fluorescence in situ hybridization with rDNA and telomeric DNA probes, C-banding, fluorochrome and silver staining. For the first time, samples from the neighbourhood of a hybrid population (i.e., Mikuni Pass population) were studied. Our results indicated a significant degree of chromosomal differentiation between P. sapporensis races when comparing the number and position of the rDNA sites, as well as the heterochromatin composition and distribution obtained by C-banding and DAPI/CMA3 staining. Telomeric signals were usually detected at the distal and/or subdistal position of the autosomes; however, some chromosome ends lacked signals, probably due to a low number of telomeric repeats. On the other hand, telomeric DNA sequences were found as interstitial telomeric repeats in some autosomes, which can trigger a variety of genome instability. B chromosomes were found in specimens belonging to both main races from nine out of 22 localities. Four types of X chromosomes in the X0/XX race were identified. It was concluded that the physical mapping of rDNA sequences and heterochromatin are useful as additional markers for understanding the phylogeographic patterns of cytogenetic differentiation in P. sapporensis populations.