The prevalence of Potato spindle tuber pospiviroid (PSTVd) infection in randomly selected potato tubers was determined via nonisotopic dot blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). In January and February of 2010, 168 seedling tubers of Solanum tuberosum, representing 27 cultivars, were received from different seedling potato producers in Turkey. Digoxigenin-labeled RNA probes of PSTVd were synthesized from the cloned PCR products and applied in dot blot hybridization to detect viroids in RNA extracts isolated from dormant potato tubers. PSTVd was found in 7 samples and 6 cultivars (Innovator, Lady Jo, Russet Burbank, Agria, Provento and Konsul). Some RNA samples, although positive by RT-PCR test, did not give a hybridization signal by dot blot assay. The complete genome of a selected isolate (PSTVd-TR) was cloned and sequenced. Primary and secondary structure analysis showed that the RNA genome of the PSTVd-TR isolate (GenBank Accession No. HQ456944) differed from other compared isolates by only a few nucleotides. The highest identity (99%) was in a PSTVd isolate that was identified in Solanum spp. in Italy (Accession No. EF459700). The infectivity of PSTVd-TR isolate was shown 2 weeks after the mechanical inoculation of sap extracts onto seedlings of tomato cultivar Joker. The PSTVd infections were proven by RT-PCR assay in symptomless tomato seedlings.