Transmission of Tomato spotted wilt virus (TSWV) is dependent on virus uptake in the midgut prior to virus movement to the salivary glands. Replication of TSWV in the alimentary canal of tobacco thrips (TT, Frankliniella fusca) and western flower thrips (WFT, F. occidentalis) was investigated by immunolocalization of the nonstructural protein (NSs) encoded by the small RNA of TSWV and fluorescence microscopy. Analysis of cohorts during development from larva to adults following virus acquisition by first instar larva indicated that virus replication followed a specific time-course pattern in the foregut, regions of the midgut, salivary glands, and ligaments between the midgut and salivary glands. Initial virus replication occurred only in epithelial cells of midgut-1 but, upon infection of muscle cells, the virus moved to the midgut-2, foregut, midgut-3, and salivary glands. The ligaments between the midgut and salivary glands appeared to be a route for virus to invade the salivary glands. No virus replication was observed in the hindgut, Malpighian tubules, or tubular salivary glands. The dynamics of TSWV replication, as measured by NSs accumulation, were similar in both TT and WFT.