Accurate identification of plant pathogens is crucial towards developing sustainable control strategies to ensure sustainable economic agricultural production. The aim of this study was to detect and characterize Potato virus Y (PVY) isolates infecting potato (Solanum tuberosum L.) in the Msinga district in the Province of KwaZulu-Natal, South Africa. Potato leaf samples exhibiting virus-like symptoms were collected from four different areas in the district. Initial detection of PVY in the leaf samples was done using triple antibody sandwich ELISA. PVY-positive samples were further tested using antibodies specific to PVY serotypes O and N. Nicotiana tabacum cv Samsun plants were individually mechanically inoculated with all 32 PVY-ELISA positive samples. Symptoms on inoculated tobacco plants were monitored over a 4-week period. They consisted of vein clearing, faint mosaic patterns, and the veinal necrosis, symptoms characteristic of PVYN, PVY(N)Wilga and PVYNTN strains. Reverse transcription-polymerase chain reaction, using primers specific to the coat protein gene of PVY, was performed as a confirmation test on total RNA of four randomly selected PVY-ELISA positive samples, each sample representing each of the four areas surveyed. Strains PVYN and PVYO were identified. The second part of the study aimed to analyse the full genome sequences of the PVY isolates A4, KD2, MOD1 and SneP3, in order to understand the evolution of the virus in Msinga. To achieve this, total RNA, extracted from tobacco leaves (N. tabacum cv Samsun) that had been inoculated with the selected four PVY isolates, was used as a template for next generation sequencing (NGS). NGS was run on Illumina HiSeq using paired-end chemistry 125 x 125bp reads. de novo assembly of the generated reads was performed. The resulting contigs were subjected to BLAST on the GenBank database in order to identify PVY genomes. The PVY isolates were aligned with closely related non-recombinant PVY sequences comprising of the following strains: PVYN, PVYO, PVYNTN, and PVYC. Recombination events were assessed using RDP4 software. Phylogenetic results revealed that PVY isolate SneP3 belonged to the PVYNTN strain while isolate PVYMOD1 to the PVY N Wilga strain group. Recombination analyses confirmed the occurrence of PVY recombinant strains in the Msinga district. The widespread presence of PVY and occurrence of recombinant strains in Msinga has serious implications on the management of PVY diseases by small-scale farmers growing potato for a livelihood. (C) 2017 Elsevier Ltd. All rights reserved.