Canola (Brassica napus) is one of the most important members of Brassicaceae family and Turnip mosaic virus is one of the most widespread viruses in canola fields all around the world. During May and April of 2018, 66 canola samples were collected from different regions of south Khorasan Province. The samples showed symptoms such as yellowing, stunning, mosaic and leaf distortion. Total RNA of samples was extracted by Dena Zist kit (Iran) and then DNA complementary was made by reverse primer. RT-PCR is done using specific primers of TuMV. A fragment with 680 bp length was amplified and sequenced in 12 canola samples that had high similarity to TuMV. Therefore, positive samples used in RT-PCR by specific primers of TuMV related to coat protein coding regions. Finally eight amplified fragments with 980 bp length were sequenced and then analyzed by Blast, MegaX, SDTv and DnaSPs softwares. Results showed that six samples of canola were infected by TuMV. Consequently, phylogenetic analysis of four non- recombinant isolates that are sequenced perfectly used in the phylogeny analysis and results showed that Iranian isolates in this study located in Asian BR phylogeny group included other Asian isolates. Nucleotide similarity between Iranian isolates ranged from 90 to 95%. Recombination analysis using RDP4 showed that two Iranian isolates (CSe55, CSe39) are recombinant and are evolved from minor and major parents. Nucleotide diversity by DnaSP showed that population of TuMV is developing in this part of Iran. This survey is the first report of TuMV in south khorasan and molecular investigation of TuMV in Canola field.