Background <p>The Epstein–Barr Virus (EBV) has a high global prevalence and is strongly associated with the pathogenesis of diverse diseases. Types 1 (T1) and 2 (T2) represent the two major EBV subtypes. However, their infectivity and pathogenic mechanisms remain unclear. We aimed to explore the differences between T1 and T2 EBV infections using the tree shrew model.</p> Methods <p>T1 and T2 EBVs were purified from the supernatant of EBV-producing cells lines B95-8 and P3HR-1, respectively cell line. Eighteen tree shrews (evenly divided by sex) were randomly assigned to three groups (<i>n</i> = 6). Each animal intravenously received 1 × 10⁸ copies of either T1 EBV, T2 EBV, or RPMI-1640 medium (vehicle control) via the femoral vein. Peripheral blood was collected at 3, 7, 14, 21, and 28 days post-inoculation (dpi) for quantitative EBV-DNA detection. peripheral blood mononuclear cells (PBMCs) were collected on 7 and 28 dpi for RNA-seq analysis, and comparisons were made with pre-infection samples to assess host transcriptional changes.</p> Results <p>EBV-DNA was detectable in the PBMCs of all six tree shrews by day 3 post-inoculation in the T1 EBV (B95-8) group, peaking at 1 × 10^4.34 copies/mg. The positivity rate for EBV infection declined over time, with one tree shrew remaining positive on day 28. In the T2 EBV (P3HR-1) group, only one of the six animals exhibited low levels of PBMCs EBV-DNA at 1 × 10^2.31 copies/mg on day 3, with no subsequent detections. Plasma EBV DNA trends were similar. Pathological examination revealed consistent LMP1 protein and EBERs expression in the spleen and lymph nodes of T1 tree shrews, with persistent plasma EBV DNA. RNA-seq revealed distinct differentially expressed genes for T1 and T2.</p> Conclusions <p>T2 EBV demonstrated significantly lower infectivity than T1 EBV in the tree shrew model. T1 infection elicits inflammatory responses, whereas T2 infection induces modest transcriptional alterations.</p>