Polymerase chain reaction linked automated sequencing was used to compare the relatedness of Brazilian (BR) potato leafroll luteovirus (PLRV) isolates. By comparing PLRV genomic sequences from Holland, Poland, Canada, Scotland, and Australia (GeneBank), three primer pairs were designed and made that would hybridize to all five isolates, and amplifying three variable regions which display distinctive nucleotide variations in each isolate. These primer pairs were used in the reverse-transcription polymerase chain reaction (RT-PCR) to analyze analogous variable regions in three PLRV isolates from around Sao Paulo State, BR (two from field potatoes grown 500 km apart, one fromDatura stramonium). Each primer pair amplified a DNA product of predicted size from the BR isolates and a Wisconsin (USA) isolate, and all PCR products were directly sequenced. Comparisons of the variable region sequences from the three Brazilian isolates to the five isolates from GenBank and the one from Wisconsin revealed that the BR isolates were approximately 99% homologous with each other, 97% with the European and Canadian isolates, and 95% with the USA and Australian isolates. These results suggest that the primer pairs utilized in this study can be used to detect PLRV (by RTPCR) for diagnostic purposes, and can further be used to differentiate between different PLRV isolates (when RTPCR is linked to automated sequencing) for epidemiological purposes.