Type III interferons (IFNλs) mediate antiviral defence at epithelial barrier tissues with limited pro-inflammatory activity, such as those found in the airways. Humans have four IFNλs: IFNλ1-4 and recent research has begun to unveil functional differences between IFNλs, particularly regarding their expression and temporal activity. Although they are all thought to signal through the same receptors, it is unclear whether they generate distinct spatial patterns of antiviral protection. To this end, a “reverse plaque assay” was established, which assessed IFNλ function during conditions of transfection-mediated non-saturating expression during infection with encephalomyocarditis virus (EMCV) or influenza A virus (IAV). Human liver and lung cells protected from cell death by IFNλs were visualised by fluorescent imaging and spatial information quantified via image analysis. Both manual and automated measurements revealed clear differences in the pattern of antiviral protection induced by IFNλ3 versus IFNλ4. IFNλ3 consistently produced dense, continuous zones of protected cells, suggestive of effective spread of signalling. In contrast, IFNλ4 produced more fragmented and spatially separated protected cell clusters, indicating less diffuse antiviral state. These patterns observed in independent of cell line or virus used. In conclusion, IFNλ3 and IFNλ4 differ not only in magnitude and kinetics of antiviral activity, but how it is spatially distributed. This suggests distinct specialisms for each IFNλ in shaping mucosal antiviral immunity, which could be exploited for development of antiviral interventions. Future work will focus on the mechanisms underlying these differences and their impact on virus host interactions relevant to transmission and pathogenesis.