Atta sexdens rubropilosa is an important leaf-cutting ant species considered as a pest in agricultural crop or reforestation areas. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) is a technique that can help us to understand the regulation and the function of a gene. However, its reliability depend on the data normalization. Different normalization strategies can be adopted for qPCR, reference genes has been cited as one of the most effective methods. It has not been identified a universal reference for all organism and experiment. In this way, the validation of reference gene is crucial step. This is the first study to evaluate reference genes for leaf-cutting ants. To this, we analyzed the expression levels of candidate reference genes (act, ef1-alpha, ef1-beta, GAPDH and rpl18) in different developmental stages (larva, pupa and worker) and tissues (head, mesosoma and worker without gaster) of A. sexdens rubropilosa. Four different algorithms (BestKeeper, geNorm, NormFinder and comparative ΔCt method) were used in statistical analysis of the stability of the genes and RefFinder was used to propose a consensus list for ranking the reference genes. Our results showed that the most suitable combinations of reference gene candidates were rpl18 and ef1-alpha for the different developmental stages and rpl18 and ef1-beta for the different tissues. In this work, we also report the obtaining from a putative acetylcholinesterase from A.sexdens rubropilosa (GenBank KY464935), which was used as a target gene to confirm the reliability of reference genes suggested.