Alfalfa (Medicago sativa L.) is one of the most important quality forages worldwide and is cultivated throughout China. Alfalfa is susceptible to a variety of viral diseases during its growth, which has caused huge amounts of commercial losses. However, the profile of the alfalfa virus in China remains ambiguous and the viruses transmitted by Odontothrips loti (Haliday), dominant insect pests in alfalfa, are also poorly understood. In the present study, virus diversity was investigated in the primary alfalfa-growing areas in China. A total of 18 alfalfa viruses were identified through RNA-sequencing (RNA-seq) and reverse transcription-polymerase chain reaction (RT-PCR). Two new plant viruses, Medicago sativa virus 1 (MsV1) and Medicago sativa luteovirus 1 (MsLV1), were detected for the first time. Another four viruses, including the Alfalfa ringspot-associated virus (ARaV), Alfalfa virus F (AVF), Alfalfa enamovirus 1 (AEV1), and Alfalfa deltaparitivirus (ADPV), were reported in China for the first time as well. Both Alfalfa mosaic virus (AMV) and Medicago sativa alphapartitivirus 2 (MsAPV2) are the dominant pathogens, with an infection incidence of 91.7–100%, and 74.4–97.2%, respectively. Additionally, O. loti with first- and second-instar nymphs were shown to acquire the AMV within 0.25 h of feeding on a virus-infected alfalfa. Transmission by thrips to healthy alfalfa plants was also demonstrated. Additionally, we clarified the dynamic changes in the AMV in pre-adult stages of O. loti, which indicated that the AMV is propagated in the nymph stage of O. loti. These findings provide valuable information for understanding the alfalfa virome, confirm the role thrips O. loti plays in alfalfa virus transmission, and improve our fundamental knowledge and management of diseases in China.