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Significance Although reverse genetics (RG) systems were first developed for animal-infecting viruses with segmented negative-stranded/ambisense RNA genomes over 20 years ago, such a system for plant viruses with segmented negative-stranded/ambisense RNA genomes had not been developed until now. Tomato spotted wilt virus (TSWV), a virus with tripartite negative-stranded/ambisense RNA genomes, is one of the most economically important plant viruses in the world. Many groups have tried to construct infectious clones for TSWV, but all have failed. Here we report the establishment of a RG system for TSWV. Infectious TSWV and derivatives carrying eGFP reporters were rescued entirely from complementary DNA (cDNA) clones. This RG system now provides a powerful platform to study the disease pathology of Tospoviruses during natural infection.