Pseudoscorpions are found in almost all terrestrial habitats. However, their uniform appearance presents a challenge for morphology-based taxonomy, which may underestimate the diversity of this order. We performed cytogenetic analyses on 11 pseudoscorpion species of the genus Chthonius C. L. Koch, 1843 from the Alps, including three subgenera: Chthonius (Chthonius) C. L. Koch, 1843, C. (Ephippiochthonius) Beier, 1930 and C. (Globochthonius) Beier, 1931 inhabiting this region. The results show that the male diploid number of chromosomes ranges from 21–35. The sex chromosome system X0 has been detected in all male specimens. The X sex chromosome is always metacentric and represents the largest chromosome in the nucleus. Achiasmatic meiosis, already known from the family Chthoniidae, was further confirmed in males of Chthonius. C-banding corroborated the localization of constitutive heterochromatin in the centromere region, which corresponds to heteropycnotic knobs on the standard chromosome preparations. Morphological types and size differentiation of chromosomes in the karyotype suggest that the main chromosomal rearrangements in the evolution of Chthonius are centric or tandem fusions resulting in a decrease in the number of chromosomes. Pericentric inversions, inducing the change of acrocentric chromosomes into biarmed chromosomes, could also be expected. Variability in chromosome morphology and number was detected in several species: C. (C.) ischnocheles (Hermann, 1804), C. (C.) raridentatus, C. (C.) rhodochelatus Hadži, 1930, and C. (C.) tenuis L. Koch, 1873. We discuss the intraspecific variability within these species and the potential existence of cryptic species.