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ABSTRACT Mice in the genusPeromyscusare abundant and geographically widespread in North America, serving as reservoirs for zoonotic pathogens, includingBorrelia burgdorferi(B. burgdorferi), the causative agent of Lyme disease, transmitted byIxodes scapularisticks. While the white‐footed mouse (Peromyscus leucopus(P. leucopus)) is the primary reservoir in the United States, the deer mouse (P. maniculatus), an ecologically similar congener, rarely transmits the pathogen to biting ticks. Understanding the factors that allow these similar species to serve as a poor and competent reservoir is critical for understanding tick‐borne disease ecology and epidemiology, especially as climate change expands the habitats where ticks can transmit pathogens. Our study investigated immunological differences between these rodent species. Specifically, we compared the expression of six immune genes (i.e., TLR‐2, IFN‐γ, IL‐6, IL‐10, GATA‐3, TGF‐β) broadly involved in bacterial recognition, elimination, and/or pathology mitigation in ear biopsies collected by the National Ecological Observatory Network (NEON) as part of their routine surveillance. A principal components analysis indicated that immune gene expression in both species varied in two dimensions: TLR2, IFN‐γ, IL‐6, and IL‐10 (comprising PC1) and TGF‐β and GATA3 (comprising PC2) expression tended to covary within individuals. However, when we analyzed expression differences of each gene singly between species,P. maniculatusexpressed more TLR2, IL‐6, and IL‐10 but less IFN‐γ and GATA3 thanP. leucopus. This immune profile could partly explain whyP. leucopusis a better reservoir for bacterial pathogens such asB. burgdorferi.