Alfalfa mosaic virus (AMV), a pathogen of a wide range of plant species, including Medicago sativa (Alfalfa) is considered as one of the most widely distributed viruses in Saudi Arabia. The molecular procedure, reverse transcription – polymerase chain reaction (RT-PCR) was developed in this study for the detection and identification of AMV in Alfalfa plants from Diyrab, Riyadh, Saudi Arabia. Alfalfa leaves were firstly screened for the presence of AMV by a standard double antibody sandwich ELISA and dot blot hybridization. AMV RNAs were easily detected in composite samples of 20 to 30 alfalfa leaves and the sequences of the coat protein (CP) gene of the isolates were determined. The primers designed on the basis of published sequences were applied for amplification of AMV RNA fragments in reverse transcription-polymerase chain reaction (RT-PCR) using infected plants with AMV. The results from this study were characterized AMV at the molecular level and their genetic relationships with other known AMV isolates were documented. Phylogenetic analysis indicated that all detected AMV isolates fell into two groups with the identified isolates from different origins. Similarity among one group ranged between (96 – 100%) and between the two groups was 42%. Evaluations showed that RT-PCR was sensitive and specific for AMV detection in Alfalfa leaf samples. The sequencing and alignment of the RT-PCR amplified fragments deposited in NCBI Gen Bank for eight different AMV strains (accession no. KF487083 – KF487090). Sequence comparison showed that these isolates of AMV shared 42% to 100% sequence similarity with seven AMV obtained from GenBank. This is the second report on the genetic variation of AMV isolates infecting alfalfa plant in Saudi Arabia. Further epidemiological studies into AMV spreading over the crops and the effect on the crop product quality are virtually needed.