Amalgaviruses are a new group of monopartite double-stranded RNA viruses identified in several plant species. Recently, genome sequences of an amalgavirus were reported from alfalfa (Medicago sativa L.), (PMID:27596539), and tentatively named Medicago sativa amalgavirus 1 (MsAV1). The MsAV1 genome was identified from alfalfa transcriptomic data released by a Chinese group (PMID:25799491). Here we present a first identification of MsAV1 by Illumina RNA-sequencing in seven out of 18 tested samples of U.S. alfalfa cultivar ZG 9830. A total of 7,910 virus reads were found among all positive samples, averaging 1,130 per sample. A complete viral genome was obtained by de novo assembly of paired-end reads (2 × 150 bp) using SPAdes or Trinity assemblers and then mapped to the available MsAV1 genome (NC_040591). Presence of the virus was experimentally confirmed by reverse-transcription polymerase chain reaction using primers derived from the de novo-assembled genome, following by cloning and sequencing of the resulting amplicons. Additionally, 5? and 3? rapid amplifications of cDNA ends were performed using a SMARTer RACE 5’/3’ kit. The U.S. isolate of MsAV1 was 100% identical to the isolate NC040591 from China at both the nucleotide and amino acid levels, indicating the same origin. This is the first experimental confirmation of MsAV1 infection in alfalfa in the United States, significant because amalgaviruses are known to be vertically transmitted through seeds.