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Plants have evolved a series of inducible or constitutive defense mechanisms in response to herbivore attack. Constitutive plant defenses are morphological and biochemical traits of the plants themselves, regardless of the presence of herbivores. We bred an alfalfa variety (Caoyuan No.4) with high thrips resistance, but the mechanisms underlying Caoyuan No.4 resistance to thrips are not well understood. To explore the constitutive defense of Caoyuan No.4, the morphological and biochemical traits associated with constitutive defense to thrips in alfalfa were analyzed using a thrips-susceptible alfalfa accession (Caoyuan No.2) as a control. The results showed that Caoyuan No.4 had thicker palisade tissue and parenchyma tissue, wider collenchyma, phloem, cambium and lignin layer, and smaller epidermal cells and stomatal aperture compared to Caoyuan No.2. Moreover, Caoyuan No.4 showed more non-glandular trichomes in both leaves and stems, but less glandular trichomes and more wax in stems. In addition, the results of the widely targeted metabolomics analysis showed that metabolites related to flavonoid, isoflavonoid, flavone and flavonol biosynthesis, as well as cysteine and methionine metabolism, differed between CaoyuanNo.2 and Caoyuan No.4. These findings shed new light on the constitutive insect defense of plants associated with physical or biochemical traits and may provide convenient markers for breeding thrips-resistant alfalfa cultivars.