Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation we used protein-binding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis reveals a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the function of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. Our results provide new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression.