[Objectives]  To identify the most appropriate reference genes on different days during the development of fifth-instar nymphs of Locusta migratoria to pave the way for relevant studies. [Methods]  Six genes were selected as candidate reference genes, including the beta actin gene (β-actin), the Elongation factor 1α gene (EF-1α), the Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), the Ribosomal protein 49 gene (RP49), the Alpha tubulin gene (α-Tubulin) and the 18S ribosome RNA gene (18S rRNA). The relative expression of these genes on different days during the development of L. migratoria was detected with quantitative real-time PCR (qPCR). The expression stability of these genes was then investigated using geNorm and Normfinder software. [Results]  Based on the results of geNorm analysis, the average expression stability (M) of the six genes was, in descending order; β-actin (0.3720)＞RP49 (0.3750)＞α-Tubulin (0.4030)＞18S rRNA (0.4270)＞EF-1α (0.4970)＞GAPDH (0.6040). The less the M value the more stable the gene expression. The software took 0.15 as a constant value for pairwise variations. The appropriate number of reference genes was 2 because V2/3=0.098＜0.15. Similar results were obtained using NormFinder software. [Conclusion]  β-actin and RP49 are the most suitable reference genes during the development of fifth-instar nymph of L. migratoria.