BackgroundSeed transmission of plant viruses can be important due to the role it plays in their dissemination to new areas and subsequent epidemics. Seed transmission largely depends on the ability of a virus to replicate in reproductive tissues and survive during the seed maturation process. It occurs through the infected embryo or mechanically through the contaminated seed coat. Alfalfa (Medicago sativaL.) is an important legume forage crop worldwide, and except for a few individual seedborne viruses infecting the crop, its seed virome is poorly known. The goal of this research was to perform initial seed screenings on alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System in order to identify pathogenic viruses and understand their potential for dissemination.MethodsFor the detection of viruses, we used high throughput sequencing combined with bioinformatic tools and reverse transcription-polymerase chain reactions.ResultsOur results suggest that, in addition to common viruses, alfalfa seeds are infected by other potentially pathogenic viral species that could be vertically transmitted to offspring.ConclusionsTo the best of our knowledge, this is the first study of the alfalfa seed virome carried out by HTS technology. This initial screening of alfalfa germplasm accessions maintained by the NPGS showed that the crop’s mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. The information gathered will be used to update germplasm distribution policies and to make decisions on the safety of distributing germplasm based on viral presence.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12985-023-02063-6.