Simple SummaryIn gene expression investigations, the first crucial step is choosing appropriate housekeeping genes. However, the choice of reference genes is not absolute but relative and varies with different experimental conditions. It is vital to note that using unvalidated or unscreened internal reference genes can lead to erroneous inferences. This study was conducted onLeptocybe invasato calculate the stability of eight housekeeping genes across various test conditions, such as sexes, somites, temperatures, diets, and pesticides. The relative expression ofHSP90at different temperature settings was evaluated to validate the results. This study aims to assist future gene expression research on this invasive species and lay the groundwork for further investigations into the gene function of this pest.AbstractLeptocybe invasa(Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression ofL. invasa’s target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes’ stability (RPS30,ACTR,18S rRNA,ACT,RPL18,GAPDH,28S rRNA, andTUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water,Eucalyptussap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied thatACTandACTRwere the most accurate when comparing sexes. For analyzing different somites,28S rRNAandRPL18were ideal; the28S rRNAandRRS30were perfect for analyzing at different temperatures. The combination ofACTandGAPDHhelped to analyze gene expression in different diets, andGAPDHand28S rRNAwere suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes fromL. invasafor precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.