Abstract: The major rDNA genes are composed of tandem repeats and are part of the nucleolus organizing regions (NORs). They are highly conserved and therefore useful in understanding the evolutionary patterns of chromosomal locations. The evolutionary dynamics of the karyotype may affect the organization of rDNA genes within chromosomes. In this study, we physically mapped 18S rDNA genes in 13 Neotropical ant species from four subfamilies using fluorescence in situ hybridization. Furthermore, a survey of published rDNA cytogenetic data for 50 additional species was performed, which allowed us to detect the evolutionary patterns of these genes in ant chromosomes. Species from the Neotropical, Palearctic, and Australian regions, comprising a total of 63 species from 19 genera within six subfamilies, were analysed. Most of the species (48 out of 63) had rDNA genes restricted to a single chromosome pair in their intrachromosomal regions. The position of rDNA genes within the chromosomes appears to hinder their dispersal throughout the genome, as translocations and ectopic recombination are uncommon in intrachromosomal regions because they can generate meiotic abnormalities. Therefore, rDNA genes restricted to a single chromosome pair seem to be a plesiomorphic feature in ants, while multiple rDNA sites, observed in distinct subfamilies, may have independent origins in different genera.