Tomato spotted wilt virus (TSWV) is a major yield-limiting pathogen of peanut in the southeastern USA. To assess viral variability, field isolates were collected from symptomatic peanut plants at three locations in Alabama. Sequence analysis identified mutations in the non-structural movement protein (NSm) and the nucleocapsid protein (N). Subcellular localization studies showed that NSm fused to GFP (NSm:GFP) localized to plasmodesmata and co-localized with callose deposits by 2days postinfiltration (dpi). By 4 dpi, NSm:GFP formed cytoplasmic aggregates, and callose deposition appeared more consistent with basal plasmodesmatal patterns. The N protein localized to the nuclear periphery and cell margins at 2 dpi and later aggregated in the cytoplasm by 4 dpi. The early callose accumulation associated with NSm expression suggests a potential plant defence response, warranting further investigation.