Cytogenetic techniques provide powerful insights on species-rich taxa–such as ants–allowing better understanding of their biodiversity. Some hints on evolutionary paths can be observed through comparative populational cytogenetics among different ant groups. In this study, the karyotype of Strumigenys louisianae Roger from the Amazon rainforest is described and showed diploid chromosome number of 26 chromosomes. This configuration intriguingly contrasts with the already described karyotype for this species from the Atlantic rainforest with only 2n = 4 chromosomes. 18S rDNA site were detected on the pericentromeric region of the long arm of a metacentric pair and co-localizing with GC-rich chromatin. Recurrent synonymizations of S. louisianae may not reflect the species status of this taxon. The karyotypic differences and the observable morphological variation between the populations of both localities corroborates the idea of a species complex within S. louisianae. The morphology of S. louisianae from the Amazonian region is similar to that from the United States, the type locality. On the other hand, specimens from the Atlantic rainforest are more similar to the junior synonym Strumigenys unidentata Mayr. This study reinforces the need of taxonomical revision in S. louisianae by means of integrative taxonomy approaches.