Viroids are the smallest pathogens composed of circular RNA, which does not encode any protein. To date, viroid infections have been reported in dicotyledonous plants. In particular, several studies on viroid-host interactions have been reported (Ding 2009). Trees in the genus Prunus, including apricot, cherry, peach, and plum, are hosts for many different viruses and several viroids, such as Apple scar skin viroid (ASSVd), Hop stunt viroid (HSVd), and Peach latent mosaic viroid (PLMVd). In Korea, PLMVd infections in peach trees have recently been reported (Jo et al. 2016); however, HSVd infections in peach trees have not been reported until now. Recently, we performed RNA-Seq to identify viruses and viroids infecting peach trees. We collected leaf samples from five popular peach cultivars—Mibaek, Janghowon, Cheonjung, Cheonhong, and Baekcheon—grown in three different regions in Korea during May 2013. We extracted total RNA from the collected leaf samples using the Fruit-mate for RNA Purification Kit (Takara, Shiga, Japan) and the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturers’ protocols. We then prepared five libraries using the TruSeq RNA Library Preparation Kit v2 (Illumina, CA, U.S.A.) according to the manufacturer’s protocol. The prepared libraries were paired-end sequenced using Illumina’s HiSEquation 2000 system (Macrogen, Seoul, Korea). For de novo transcriptome assembly, we used the Trinity program (Haas et al. 2013). A BLAST search was conducted using assembled transcriptome data against a viral genome reference database from NCBI using a standalone BLAST program. Of the identified virus-associated contigs, we deleted endogenous virus-like contigs and identified only virus-associated contigs. We found that transcriptomes from five peach cultivars contained HSVd-associated contigs. To confirm HSVd infection in the five peach cultivars, we carried out reverse-transcription (RT)-PCR using a pair of HSVd-specific primers, VP-19 5′-GCCCCGGGGCTCCTTTCTCAGGTAAG-3′ (position 85-60) and VP-20 5′-CCCGGGGCAACTCTTCTCAGAATCC-3′ (position 78-102) (Amari et al. 2001). The amplified PCR products were cloned into the pGEM-T-Easy Vector (Promega, Wisconsin, U.S.A.) followed by Sanger sequencing (accession numbers KX371905 to 09). The BLAST results using amplified PCR products confirmed the HSVd infection. In addition, we examined the infection of ACLSV and PLMVd by virus-specific primers. Again, we found that all five cultivars were coinfected by at least ACLSV and PLMVd. We examined the disease symptoms of five peach cultivars from 2014 to 2015; however, we did not find any severe disease symptoms caused by the coinfection of viruses and viroids. To the best of our knowledge, our study represents the first report of HSVd infection in peach trees in Korea. © 2016, American Phytopathological Society. All rights reserved.