Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows large-scale analysis of very small changes in gene expression. For the calculation of gene expression, such as the delta-delta Ct method, different PCR primer efficiencies (E) may affect the result, as PCR primer yields are assumed to be comparable for the gene of interest and housekeeping gene. Therefore, identification of a suitable reference gene for data normalization is an important step in the development of qPCR assays. Furthermore, accurate and reliable results depend on the use of stable reference genes for normalization. The aim of the current study is the identification and validation of a set of six housekeeping genes (GADPH, RPS18, α-TUB, EF1α, ArgK, and ACTB) in cockroach species Nauphoeta cinerea adults using five different algorithms (ΔCt method, Bestkeeper, geNorm, Normfinder and RefFinder) to evaluate the stability of selected reference genes expression. Our results show that α-Tub use provides accurate normalization of gene expression levels in N. cinerea adults. In addition, since the GADPH is selected as the second most stable reference gene, GADPH can be also used for transcript analysis N. cinerea adults. Our study also showed that ACTB (β-actin) should not be used for normalizing transcript levels when examining N. cinerea adults. Additionally, validation studies for reference genes in cockroaches are very few (only one) in the literature. Therefore, the results highlight the need for validation of reference genes under biotic and abiotic conditions in q-RT-PCR studies in cockroaches.