© 2020 Parlar Scientific Publications. All rights reserved.Survey studies were carried out in in 2018 in Adiyaman, Turkey, where tobacco is traditionally produced, to detect the viruses that infect tobacco including Tobacco mosaic tobamovirus (TMV), Cucumber mosaic cucumovirus (CMV), Potato potyvirus Y (PVY), Tobacco vein banding mosaic potyvirus (TVBMV), and Tobacco etch potyvirus (TEV). Accordingly, 450 fresh tobacco leaves were simultaneously analyzed by the multiplex RT-PCR method for the presence of tobacco viruses. As a result of molecular analysis, CMV (8.8%), TMV (5.5%), and TMV + CMV mixed infection (0.2%) were detected in 40 of the tobacco samples. TEV, PVY, and TVMBV viruses were not detected in any of the samples. The overall infection incidence of tobacco viruses in Adiyaman province was found to be 14.4%. Two isolates were randomly selected from each detected virus and their coat protein (CP) genes were cloned into the prokaryotic cloning vector and transformed into E. coli. Recombinant plasmids obtained from positive clones were sequenced by Next Generation Sequencing (NGS) system to reveal the viral (CP) sequence. Nucleic acid sequence similarity analyses revealed that the TMV Adiyaman isolates showed similarity in the range of 74.17-99.38% and the CMV Adiyaman isolates in the range of 75.61-99.54%. In the phylogenetic analyis using (CP) sequences, tobacco-CMV isolates were found to be in phylogenetic affinity with the Greece (AY450854) and Japan (AB448696) isolates, while both tobacco-TMV isolates showed a phylogenetic affinity with the Serbia isolate (GQ340671). According to the current literature, this is the first molecular survey study on tobacco viruses in Adiyaman and Turkey. With the present study, the molecular char acterization of the isolates selected with the presence of CMV and TMV in the tobacco plant has been re¬ ported for the first time.