Alfalfa ( Medicago sativae L.) as a perennial herbaceous legume is the most important forage crops in the world. Aphid-borne viruses infecting alfalfa such as Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Peanut stunt virus (PSV) and Bean leaf roll virus (BLRV) are the most important viruses in decreasing alfalfa yield. Despite many reports of aphid-borne viruses incidence in alfalfa around the world, there is no information on aphid-borne viruses incidence in central region of Iran. To detect AMV, CMV, PSV and BLRV, in 1394 and 1395, alfalfa samples showing symptoms such as leaf malformation, little leaf, dwarfing, interveinal and vein yellowing were collected from alfalfa fields in Isfahan and Chaharmahal Bakhtiari provinces. Total RNA was extracted from leaf samples and used as template in RT-PCR with specific primer pairs AMV-5pr/AMV-3pr (901 bp), CMV-MP5/CMV-MP3 (843 bp), PSV-F8/PSV-R8 (932 bp) and BLRV-F1/BLRV-R2 (621 bp) for detection of AMV, CMV, PSV and BLRV, respectively. The specific bands of AMV, PSV and BLRV were amplified in several samples in RT-PCR. However, no CMV specific band was amplified in collected samples. The PCR products of some AMV, PSV and BLRV infected samples were sequenced. The BLAST analysis confirmed the RT-PCR results. Out of 173 collected samples, 69 samples were infected with AMV, three samples were infected with PSV, five samples were infected with BLRV, 66 samples had mixed infection with AMV/PSV, 16 samples had mixed infection with AMV/BLRV, one sample had mixed infection with PSV/BLRV and 13 samples had mixed infection with AMV/PSV/BLRV. For molecular comparisons seven PSV isolates (D1, D14, D17, F1C1, Mo22, M8 and LoA4) and nine BLRV isolates (B1, M3, D5, H2, Fa5, DaC, DaE5, ShB7 and F1D1) collected from different geographical regions were sequenced. Multiple alignments of nucleotide sequence of coat protein gene of Isfahan and Chaharmahal Bakhtiari PSV and BLRV isolates and those of other isolates available at GenBank were carried out by CLC sequencing viewer 7.7 and DNAMAN 4 softwares. The results showed Isfahan and Chaharmahal Bakhtiari PSV isolates shared 97.79% identity with each other. The highest identity (94.4%) was found between Mo22 isolate (Mobarakeh) and W isolate of United States (Accession No. U31366). D14 and D17 isolates (Dastgerd) and S isolate of China (Accession No. AJ222803) showed the lowest identity (74.2%). Phylogenetic analysis by Neighbor-joining method with 10000 bootstrap replicates using MEGA 5 software revealed that Isfahan and Chaharmahal Bakhtiari PSV isolates clustered together with GenBank isolates of PSV subgroup II. Multiple alignment of BLRV isolates showed that Isfahan and Chaharmahal Bakhtiari BLRV isolates shared 99.4% identity to each other. D5 (Dastgerd) and isolates from Argentina (Accession No. KR261610) and United States (Accession No. 003369) had the highest identity (96.3%) and Fa5 isolate (Farokhshahr) and WA isolate from United States (Accession. No. HM439776) had the lowest identity (94.4%). In phylogenetic tree drawn by Neighbor-joining method with 10000 bootstrap replicates using MEGA 5 software, Isfahan and Chaharmahal Bakhtiari BLRV isolates were grouped in a distinct cluster and separated from other isolates of BLRV available at GenBank. To determine viral infection in alfalfa seeds, total RNA was extracted from 13 alfalfa plants with seed pods showing viral symptoms and subjected to RT-PCR using AMV, CMV, PSV and BLRV specific primer pairs. The results showed all alfalfa plants are only infected with AMV. The alfalfa seeds were allowed to germinate in Petri dishes containing wet filter papers. Three days after germination, total RNA was extracted from seedlings and RT-PCR was carried out. The results showed the presence of AMV in seeds of 11 out of 13 alfalfa plants. It seems that aphid-borne viruses infecting alfalfa in decreasing order of incidence in alfalfa fields of Isfahan and Chaharmahal Bakhtiari provinces are AMV, BLRV and PSV (subgroup II), whereas CMV has no importance. In addition, there is no distinct relationship between virus species and symptom type in infected plants. Keywords : Alfalfa mosaic virus , Peanut stunt virus , Bean leaf roll virus , RT-PCR, Coat protein gene, Seed, Alfalfa