The long-term effects of doxorubicin (DOX) chemotherapy on thymic immune function in childhood cancer survivors remain inadequately understood. This study explores the immediate and delayed impacts of low-dose DOX on thymic immune populations using a juvenile mouse model. Male mice received intraperitoneal DOX injections (4 mg/kg/week) for three weeks, with evaluations performed at one- and five-weeks post treatment. Thymic samples were collected and analyzed using multi-parameter flow cytometry to assess changes in immune cell composition and phenotype. Additionally, real-time polymerase chain reaction (RT-PCR) was employed to measure gene expression of cytokines and senescence markers. One week after DOX administration, significant thymic atrophy was evident. While mature CD3CD4 T-cell frequency remained unchanged, CD3CD8 T-cells significantly increased, suggesting differential effects on T-cell subsets. PD1 expression increased across naïve and memory CD4 T-cell subsets, suggesting activation or exhaustion. Additionally, Ki67 expression was elevated in naïve and memory CD8 T-cells, indicating enhanced proliferation. Gene expression analysis revealed upregulation of Foxn1, Pax1, Ifnγ, and Il7 whereas Il6 and Il17 were downregulated. Furthermore, Cdkn1a (p21) expression was elevated, suggesting immune dysregulation and early immunosenescence. At five weeks, thymic weight rebounded; however, T-cell subsets displayed persistent perturbations. Central memory and effector memory CD4 T-cells were reduced, while naïve CD4 T-cells showed increased Ki67 expression. In contrast, CD8 T-cells subsets remained largely unchanged, except for a decrease in central memory cells. Although expression of thymus-related genes was normalized, p21 expression remained elevated, suggesting lingering immunosenescence. These findings highlight the complex effects of DOX, including acute thymic atrophy due to T-cell depletion, and a delayed recovery with persistent immunosenescence, underscoring the need for strategies to preserve immune function in childhood cancer survivors.