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Background: MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation that down-regulate target genes by mRNA degradation or translational repression.Evidence is increasing for their crucial roles during plant development.ldentification of miRNAs at the global genome-level by high-throughput sequencing is essential to functionally characterize miRNAs in plants.Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial forage legumes worldwide.Fall dormancy is an adaptive character related to the biomass production and winter survival in alfalfa.However, little is known about miRNA-mediated developmental regulation of fall dormancy in alfalfa.Here, we applied high-throughput sRNA sequencing and genome-wide mining to identify miRNAs that were responsive to fall dormancy.