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Abstract Background Deer mice (Peromyscus maniculatus) are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV), the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT), Th2 cells (GATA-3, STAT6, IL-4, IL-5) and regulatory T cells (Fox-p3, IL-10, TGFβ1). These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.