Plant viruses may behave differently in glasshouse than in nature, especially those that are naturally transmitted by vector. Biological assays, as the oldest and still used virus detection methods, involve inoculation of host plant and monitoring symptoms in glass- or green-house. While maintaining Peanut stunt virus (PSV) in glasshouse we came across with symptom attenuation of PSV strain B (PSV-B) in Nicotiana benthamiana after eight successive passages. To determine if any changes have occurred in the PSV-B genome, the virus particles were purified from the plant and subjected to RNA extraction with phenol/ chloroform to remove the virus coat protein (CP), and followed by precipitation with ethanol to purify the virus RNAs. Then, electrophoresis of the virus RNAs on agarose gel showed that the RNA3 component of the PSV-B isolate was longer than that in the original reference PSV-B, suggesting occurrence of insertion(s) in the RNA3. To confirm this observation, the full length cDNA corresponding to the RNA3 was synthesized by RT-PCR with the use of the 5’ and 3’ terminal primers which were designed according to sequences of the PSV RNA3 terminal ends (accession PSU31366). The RT-PCR product was subjected to dideoxy cycle sequencing and the sequence data was compared with the corresponding PSV RNA3 sequences. This revealed occurrence of a 183- nucleotide repeat at the 3’ untranslated region of the RNA3 and suggested an association between this mutation (repeat) and the symptom attenuation. This emphasizes that in glasshouse studies, sequential passages of a virus can lead to emergence of mutants. This observation also has implication as to plant protection because the mutated variant may be used in cross protection.