Bothriurus Peters, 1861 is one of the most diverse genera within the family Bothriuridae. However, to date, only five species have been analyzed using a cytogenetic approach. In this study, for the first time, two populations of Bothriurus asper Pocock, 1893 and nine populations of Bothriurus rochai Mello-Leitão, 1932, two species from northeastern Brazil, were analyzed with respect to diploid number, chromosomal behavior during meiosis, and the localization of heterochromatin and nucleolus organizer regions (NORs). For B. asper, a diploid number of 2n = 30 was recorded in geographically distant populations, whereas B. rochai exhibited intraspecific variation in diploid number (2n = 16 and 2n = 18), representing the lowest diploid numbers ever reported for the family Bothriuridae. Despite the variability in diploid number, the number and localization of NORs remained stable among the populations of B. rochai. When comparing heterochromatin patterns between the two species, larger blocks of constitutive heterochromatin were observed in B. asper than in B. rochai. Variation in the amount of heterochromatin among populations of B. rochai was also observed; in this case, the population with the lowest amount of heterochromatin also exhibited the greatest variation in post-pachytene cell configurations. This is the first study to cytogenetically analyze multiple populations of species within the genus Bothriurus, and it significantly expands the karyotypic information available for scorpions with monocentric chromosomes.