Non-target-site based resistance to herbicides (NTSR) is complex, widespread, unpredictable and can confer resistance to a range of herbicides with different modes of action. Thus, it is the major threat to grass weed chemical control. NTSR to ACCase and to ALS inhibitors is widespread in France, especially among Poaceae as black-grass (Alopecurus myosuroides) and ray-grass (Lolium ssp). Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. NTSR is endowed by a subset of the genes involved in herbicide stress response. Therefore, differences in gene expression between resistant and sensitive plants can be at the root of NTSR and a transcriptomics-based approach is expected to identify NTSR candidate genes. Currently, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is the most accurate tool to determine differences in gene expression. Using this method, candidate genes identified by RNA sequencing in blackgrass and ray-grass sprayed with ACCase and ALS inhibitors were analysed, respectively. Expression profiles observed after RNA-sequencing and differences of expression between resistant and sensitive plants were confirmed by RT-qPCR for all genes studied. We enlarged results to other plants and populations of blackgrass and ray-grass, and noticed that the differences of expression observed in plants used for RNA-sequencing were generally not observed anymore. We concluded that no clear cut exists between the resistance and the expression of one unique gene, and that NTSR should be explained by the over-expression of several genes simultaneously.