Leaf-cutting ants are among the New World's most conspicuous and studied ant species due to their notable ecological and economic roles. Cytogenetic studies carried out in Atta show remarkable karyotype conservation among the species. We performed classical cytogenetics and physical mapping of repetitive sequences in the leaf-cutting ant Atta cephalotes (Linnaeus, 1758), the type species of the genus. Our goal was to test the karyotype conservation in Atta and to understand the genomic organization and diversity regarding repetitive sequences in leaf-cutting ants. Atta cephalotes showed 2 n = 22 (18m + 2sm + 2st) chromosomes. The heterochromatin followed a centromeric pattern, and the GC-rich regions and 18S rDNA clusters were co-located interstitially in the 4th metacentric pair. These cytogenetic characteristics were observed in other Atta species that had previously been studied, confirming the karyotype conservation in Atta. Evolutionary implications regarding the conservation of the chromosome number in leaf-cutting ants are discussed. Telomeric motif (TTAGG) n was detected in A. cephalotes as observed in other ants. Five out of the 11 microsatellites showed a scattered distribution exclusively on euchromatic areas of the chromosomes. Repetitive sequences mapped on the chromosomes of A. cephalotes are the first insights into genomic organization and diversity in leaf-cutting ants, useful in further comparative studies.