Leaf-cutting ants are considered the most important herbivores in terrestrial environments throughout the Neotropics. Amoimyrmex Cristiano, Cardoso, & Sandoval, 2020 is the sister clade of the remaining leaf-cutting ants from the genera Atta and Acromyrmex. Amoimyrmex striatus was the only species cytogenetically studied within the genus and shares the same chromosomal number as Atta, bearing 22 chromosomes, whereas Acromyrmex bears 38 chromosomes, with the exception of the social parasite Acromyrmex ameliae (2n = 36). Our objective here was to cytogenetically analyze the species of Amoimyrmex bruchi and Amoimyrmex silvestrii, as well as to describe the karyotype of these sister species, using an integrative approach using classical and molecular cytogenetics. We aimed to characterize the cytogenetic markers that contribute to the systematics and taxonomy of the genus. Our results showed that the karyotypes of these two species are very similar, with an identical chromosome number (2n = 22), chromosome morphology (2K = 20m + 2sm), and location of 18S rDNA and telomeric repeat TTAGG on the chromosomes. However, the microsatellite probe GA(15) showed variation across the species and populations studied. We suggest that both species diverged relatively recently and are unmistakably sisters because of the many shared characteristics, including the highly conserved karyotypes.