<jats:title>Abstract</jats:title><jats:p>The cytogenetic characteristics of the grasshopper <jats:italic>Podisma sapporensis</jats:italic> (two races 2n = 23♂ X0/XX and 2n = 22♂ neo-XY/neo-XX) were analysed through fluorescence <jats:italic>in situ</jats:italic> 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 <jats:italic>P. sapporensis</jats:italic> 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/CMA<jats:sub>3</jats:sub> 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 <jats:italic>P. sapporensis</jats:italic> populations.</jats:p>