Biological characteristics of 11 Potato virus S (PVS) isolates from three cultivated potato species (Solanum spp.) growing in five Andean countries and 1 from Scotland differed in virulence depending on isolate and host species. Nine isolates infected Chenopodium quinoa systemically but two others and the Scottish isolate remained restricted to inoculated leaves; therefore, they belonged to biologically defined strains PVSA and PVSO, respectively. When nine wild potato species were inoculated, most developed symptomless systemic infection but Solanum megistacrolobum developed systemic hypersensitive resistance (SHR) with one PVSO and two PVSA isolates. Andean potato cultivars developed mostly asymptomatic primary infection but predominantly symptomatic secondary infection. In both wild and cultivated potato plants, PVSA and PVSO elicited similar foliage symptoms. Following graft inoculation, all except two PVSO isolates were detected in partially PVS-resistant cultivar Saco, while clone Snec 66/139-19 developed SHR with two isolates each of PVSA and PVSO. Myzus persicae transmitted all nine PVSA isolates but none of the three PVSO isolates. All 12 isolates were transmitted by plant-to-plant contact. In infective sap, all isolates had thermal inactivation points of 55 to 60°C. Longevities in vitro were 25 to 40 days with six PVSA isolates but less than 21 days for the three PVSO isolates. Dilution end points were 10−3 for two PVSO isolates but 10−4 to 10−6 with the other isolates. Complete new genome sequences were obtained from seven Andean PVS isolates; seven isolates from Africa, Australia, or Europe; and single isolates from S. muricatum and Arracacia xanthorhiza. These 17 new genomes and 23 from GenBank provided 40 unique sequences; however, 5 from Eurasia were recombinants. Phylogenetic analysis of the 35 nonrecombinants revealed three major lineages, two predominantly South American (SA) and evenly branched and one non-SA with a single long basal branch and many distal subdivisions. Using least squares dating and nucleotide sequences, the two nodes of the basal PVS trifurcation were dated at 1079 and 1055 Common Era (CE), the three midphylogeny nodes of the SA lineages at 1352, 1487, and 1537 CE, and the basal node to the non-SA lineage at 1837 CE. The Potato rough dwarf virus/Potato virus P (PVS/PRDV/PVP) cluster was sister to PVS and diverged 5,000 to 7,000 years ago. The non-SA PVS lineage contained 18 of 19 isolates from S. tuberosum subsp. tuberosum but the two SA lineages contained 6 from S. tuberosum subsp. andigena, 4 from S. phureja, 3 from S. tuberosum subsp. tuberosum, and 1 each from S. muricatum, S. curtilobum, and A. xanthorrhiza. This suggests that a potato-infecting proto-PVS/PRDV/PVP emerged in South America at least 5,000 years ago, became endemic, and diverged into a range of local Solanum spp. and other species, and one early lineage spread worldwide in potato. Preventing establishment of the SA lineages is advised for all countries still without them.