A bacilliform virus, named Potato yellowing virus (PYV), causing chlorosis of leaves was reported in 1992 in potato (Solanum tuberosum) fields in Peru (1) and symptomless wild potatoes (S. fernandezianum) in Chile (4). PYV is reported as an alfamo-like virus (1) (family Bromoviridae) but no sequence information is available for this virus, making its taxonomic position inside the Bromoviridae uncertain (currently this family is organized into five genera: Alfamovirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus). Herein we report the presence of PYV in native potatoes (Solanum phureja) collected from Ecuador where the crop constitutes an important source of income in rural communities. Forty accessions of S. phureja collected in Ecuador in June 1986 and maintained in vitro at the International Potato Center (CIP) germplasm bank were analyzed by double-antibody sandwich (DAS)-ELISA with antiserum raised against a Peruvian isolate of PYV (1). PYV was detected in six accessions (15% of the material) corresponding to cultivars Chaucha Tomate and Chaucha Blanca (from the province of Cañar), Chaucha Negra Ojona and Chaucha Amarilla (Loja Province), and Cuica and Chaucha (Azuay Province). Mechanical inoculation of the indicator plant Physalis floridiana with leaf extracts of these six plants, a PYV isolate from Peru (1) (positive control), and an additional four plants testing negative for PYV (negative controls) induced symptoms of mosaic and leaf deformation only with the six clones from Ecuador and the PYV isolate from Peru. To further confirm the presence of the virus, we used universal PCR primers designed for the Bromoviridae (Ilar1F5: 5′-GCNGGWTGYGGDAARWCNAC-3′ and Ilar1R7: 5′-AMDGGWAYYTGYTYNGTRTCACC-3′) that target the helicase motif (RNA1) (3). Total RNA was extracted from 200 mg of leaf material (from potato and mechanically inoculated P. floridiana) using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer's instructions and cDNA was synthesized using random hexamer primers. We obtained a reverse transcription-PCR amplified band only from samples that were DAS-ELISA positive to PYV. To identify the virus at the genus level, we cloned the PCR fragments (265 nucleotides) from four of the samples from Ecuador and the Peruvian isolate into plasmid vectors (pGEM-T Easy Vector cloning system; Promega, Madison, WI) for Sanger sequencing (Macrogen, Seoul, Korea). Phylogenetic analysis grouped PYV sequences with those of the genus Ilarvirus. Among the ilarviruses, Fragaria chiloensis latent virus (2) was the closest relative of PYV, with which it shares 77% nucleotide and 85% amino acid sequence identity. PYV isolates from Ecuador split into two different variants (91% identity) that shared 93% nucleotide and 99% amino acid sequence identity with the Peruvian isolate. Collectively, the data suggest that PYV is a distinct ilarvirus and that it is more widely spread among South American potatoes than previously suggested. The GenBank Accession Numbers for the sequences described are: HQ141053 (Loja1), HQ141054 (Azuay), HQ141055 (Cañar), and HQ141056 (Loja2) for the isolates from Ecuador and HQ141057 (PYV-Cañete) for the isolate from Peru. References: (1) S. Fuentes and U. Jayasinghe. Fitopatología 28:22, 1993. (2) I. E. Tzanetakis and R. R. Martin. Virus Res. 112:32, 2005. (3) M. Untiveros et al. J. Virol. Methods 165:97, 2010. (4) J. P. T. Valkonen et al. Potato Res. 35:411, 1992.