Background: Thrips (Thysanoptera: Thripidae) are major insect pests on alfalfa and result in decreased plant nutrients and growth, low yields and even plant death. In our previous studies, an alfalfa variety (Caoyuan No.4) with high thrips resistance was bred through consecutive field recurrent selection. In order to better understand the genetic and molecular mechanisms of thrips resistance in Caoyuan No.4, RNA-Sequencing was employed using the thrips-resistant alfalfa accession (Caoyuan No.4) and a thrips-susceptible alfalfa accession (Caoyuan No.2), each with and without thrips infestation.Results: There were 851 genes constitutively upregulated and 434 genes downregulated in Caoyuan No.4 compared to Caoyuan No.2 without thrips infestation. The upregulated genes were mainly involved in primary metabolism such as energy metabolism and carbohydrate metabolism, lipid metabolism and certain secondary metabolites, while the downregulated genes were mainly related to plant-pathogen interaction. In addition, very few DEGs (only 13) were detected in Caoyuan No.4 after thrips stress, but a total of 3326 contigs DEGs were detected in Caoyuan No.2 after thrips stress. The upregulated genes in Caoyuan No.2 after stress were mainly involved in isoflavonoid biosynthesis, proteasome, amino sugar and nucleotide sugar metabolism, flavonoid biosynthesis as well as plant-pathogen interaction. Moreover, 117 genes that were shared in both the S_CK vs S_T group and S_CK vs R_CK group were divided into 6 clusters, which are mainly involved in secondary metabolism, fatty acid metabolism, amino acid metabolism, rust resistance kinase, WRKY transcription factor and nodule lectin.Conclusion: Both constitutive defensive genes and potential induced defensive genes were detected in the defense of Caoyuan No.4. That two distinct kinds of defensive genes — constitutive defensive genes and induced defensive genes — can be simultaneously activated and thus potentially enhance plant protection against insects attacks is a significant finding for plant resistance breeders.