In Brazil, Potato virus Y (PVY) currently presents a significant problem for potato production, reducing tuber yield and quality. Recombinant tuber necrotic isolates of PVY had been reported to occur in the country but no systematic study of the PVY isolate diversity was conducted thus far. Here, a panel of 36 PVY isolates, randomly collected in Brazil from potato between 1985 and 2009, was subjected to a systematic molecular and serological typing using reverse-transcription polymerase chain reaction and a series of PVYO- and PVYN-specific monoclonal antibodies. The data collected were combined with biological characterization of the same isolates in tobacco. Of the 36 isolates tested, 3 were typed as PVYO, 10 as PVYN:O/N-Wi, 21 as PVYNTN, and 2 as “unusual” or inconclusive. Of the 10 isolates from the recombinant PVYN:O/N-Wi strain group, 1 isolate, MAF-VOY, was found to have an unusual serological profile identical to the nonrecombinant PVYO-O5 strain group. The 21 tested PVYNTN isolates included 1 isolate that did not induce vein necrosis in tobacco and 2 isolates with an unusual serological profile (i.e., displaying negative reactivity to one commercial PVYN-specific monoclonal antibody). Whole genome sequences were determined for four PVY isolates from Brazil, representing PVYO, PVYNTN, and PVYN-Wi strains. The genome of the MAF-VOY isolate was found to be recombinant, having characteristic N-Wi structure with two recombinant junctions and carrying a single mutation in the capsid protein at position 98, which led to an unusual O5 serological reactivity. Taken together, the data obtained suggest that the two recombinant strains, PVYNTN and PVYN:O/N-Wi, now are apparently dominant in the Brazilian potato crop. The data also suggest that recombinant isolates in Brazil often have unusual serological reactivity which may hamper their correct identification by conventional typing based on enzyme-linked immunosorbent assay.