Tomato spotted wilt virus (TSWV)-infected Capsicum annuum plants were collected from open fields during June to July in 2010. The TSWV isolates were designated as Gneung, Ghang-Kjj, Gchang-Njc, Ghae, and Pap. The nucleotide sequence of the nucleocapsid protein (N) and movement protein (NSm) of the five isolates was determined. The pathogenicity of the five isolates was determined on 14 C. annuum cultivars two times by using mechanical inoculation. The five isolates induced different response: Both Gneung and Gchang-Kjj did not infect any of the cultivars in the 2nd trial, while Gchang-Njc, Ghae and Pap infected 11, 6 and 13 of 14 cultivars, respectively. The five isolates also were tested on Solanum lycopersicum breeding line TGC09-71 and three Nicotiana species. S. lycopersicum showed a similar response to the five isolates as did C. annuum. Both Gchang-Njc and Ghae infected systemically all three Nicotiana species tested. While both Pap and Gneung did not infect any of the Nicotiana species tested. In conclusion, five TSWV isolates induced different infection spectra in C. annuum cultivars, Nicotiana species and an S. lycopersicum breeding line. Amino acid sequence analysis of the NSm gene could not support or explain the different infection spectra of the five isolates. This study indicated that various isolates must be used as virus inocula for evaluation of C. annuum and S. lycopersicum cultivars in breeding programs for TSWV resistance.