A putative virus-induced disorder showing mottling and deformation on leaves and fruits of sweet pepper was observed in Taiwan in 2009. Crude sap of symptomatic sweet pepper leaves reacted positively in enzyme-linked immunosorbent assay (ELISA) with antisera to tospoviruses in the Watermelon silver mottle virus (WSMoV) serogroup. A virus culture, TwPep3, isolated from the symptomatic sweet pepper revealed isometric virions measuring about 80–100 nm in diameter via electron microscopy. Seedlings of sweet pepper inoculated with TwPep3 showed similar symptoms as those observed in the field. A 0.9-kb viral genome of isolate TwPep3 could be amplified using degenerate primers for L RNA conserved region of tospoviruses but not using those of tobamoviruses, potyviruses and Cucumber mosaic virus. The partial L RNA sequence of isolate TwPep3 (KF383955) shared 75.1% and 82.4% nucleotide identities with those of WSMoV and Tomato necrotic ringspot virus (TNRV), respectively. To determine the taxonomic relationship of isolate TwPep3, the full length of S RNA (KF383956) was sequenced. Sequence analyses showed that the nucleocapsid (N) gene of TwPep3 shared 82–83% and 87.9–89% nucleotide and amino acid sequence identities, respectively, with those of five isolates of TNRV, the most closely related tospovirus. Moreover, the NSs proteins and S RNAs of TwPep3 and TNRV shared only 78.7% and 77.6% amino acid and nucleotide sequence identities, respectively. Based on our results, isolate TwPep3 should be considered as a new tospovirus and we propose the name Pepper chlorotic spot virus (PCSV). A specific primer pair designed in the N gene was successfully used in reverse transcription-polymerase chain reaction for PCSV diagnostic work.