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Brassica carinata (BBCC, 2n = 34) is commonly known as Ethiopian mustard, Abyssinian mustard, or carinata. Its excellent agronomic traits, including resistance to biotic and abiotic stresses, make it a potential genetic donor for interspecific hybridization. Myzus persicae (green peach aphid, GPA) is one of the most harmful pests of Brassica crops, significantly effecting the yield and quality. However, few aphid-resistant Brassica crop germplasms have been utilized in breeding practices, while the underlying biochemical basis of aphid resistance still remains poorly understood. In this study, we examined the genetic diversity of 75 B. carinata accessions and some plant characteristics that potentially contribute to GPA resistance. Initially, the morphological characterization showed abundant diversity in the phenotypic traits, with the dendrogram indicating that the genetic variation of the 75 accessions ranged from 0.66 to 0.98. A population structure analysis revealed that these accessions could be grouped into two main subpopulations and one admixed group, with the majority of accessions (86.67%) clustering in one subpopulation. Subsequently, there were three GPA-resistant B. carinata accessions, BC13, BC47, and BC51. The electrical penetration graph (EPG) assay detected resistance factors in the leaf mesophyll tissue and xylem. The result demonstrated that the Ethiopian mustard accessions were susceptible when the phloem probing time, the first probe time, and the G-wave time were 20.51-32.51 min, 26.36-55.54 s, and 36.18-47.84 min, respectively. In contrast, resistance of the Ethiopian mustard accessions was observed with the phloem probing time, the first probe time, and G-wave time of 41.18-70.78 min, 181.07-365.85 s, and 18.03-26.37 min, respectively. In addition, the epidermal characters, leaf anatomical structure, glucosinolate composition, defense-related enzyme activities, and callose deposition were compared between the resistant and susceptible accessions. GPA-resistant accessions had denser longitudinal leaf structure, higher wax content on the leaf surface, higher indole glucosinolate level, increased polyphenol oxidase (PPO) activity, and faster callose deposition than the susceptible accessions. This study validates that inherent physical and chemical barriers are evidently crucial factors in the resistance against GPA infestation. This study not only provide new insights into the biochemical basis of GPA resistance but also highlights the GPA-resistant B. carinata germplasm resources for the future accurate genetic improvement of Brassica crops.