Tobacco mosaic virus (TMV) is one of the devastating plant viruses in the world that infects more than 200 plant species. Movement protein plays a supportive role in the movement of other plant viruses, and viral coat protein is highly expressed in infected plants and affects replication and movements of TMV. In order to investigate genetic variation in the terminal fragment sequence in Iranian TMV isolate, viral RNA was extracted from the infected tobacco leaves and cDNA was constructed using a specific antisense primer, PSh60-R1. The coding sequence of movement protein and coat protein was amplified and bi-directionally sequenced using PSH63-F/R primers. The results from sequencing were analyzed by Vector NTi software, and the nucleotide sequence was aligned with other TMV isolates of Iran using MEGA 5 and CLC Main Workbenches. Based on the results, the comparison between sequences of movement protein from TMV-ir and movement protein sequence of TMV-U1 reveals six mutations. Also, nucleotide comparison reveals that there are two mutations between coat protein sequence of TMV-ir and TMV-U1. Based on sequencing results, there is the minimum nucleotide distance between TMV-ir and TMV-U1 and maximum distance between isolates of Iran (Br50, TO79, TO32, TA, PU1, TM, G42, TV). Based on the results, we report a new isolate of TMV from Iran that genetically distinct with other Iranian isolates. These results provide good knowledge that could be valuable to designing detection kits and plant breeding programs.

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