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Fast identification of pesticide residue in lettuce leaves plays a key role in the test of food safety. However, there are a few studies on the mechanism of pesticide on the internal structure of lettuce. In order to further study the effects of dimethoate on lettuce microstructure, the lettuce at rosette stage was used as the experimental object. Besides, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to detect the microstructure of lettuce leaves. It was found that the microstructure of the lettuce leaves was changed obviously with the increase of the concentration of dimethoate pesticides sprayed. Moreover, the ratio of length to width and density of stomata in leaves were decreased, leaf thickness was decreased, osmiophilic granules were increased and the starch granules were decreased. In the wavelength range of 897~1332nm, spraying dimethoate lettuce spectral reflectance was inversely proportional to the concentration of dimethoate. However, in the wavelength range of 1703~1754nm, spraying dimethoate lettuce reflectance was proportional to the concentration of dimethoate. In addition, dimethoate pesticide spraying with different concentrations resulted in different textures of lettuce leaves. The results showed that dimethoate produced a huge change on lettuce microstructure, which caused great changes of the image information of lettuce leaves. Study on the microstructure of lettuce leaves sprayed with different concentrations of dimethoate pesticide provided the basis for the study on the mechanism of HSI technology to detect the different concentrations of pesticide residues in lettuce. Therefore, hyperspectral image detection technology can effectively realize the quantitative detection of pesticide residues on the surface of lettuce leaves.