MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in development and stress response in plants by negatively regulating gene expression post-transcriptionally. Medicago truncatula has been used as a model plant to study functional genomics of legume plants. It has also been widely used to functionally study miRNAs. Identification of miRNAs at the whole-genome level is essential for functional characterization of miRNAs in plants. High-throughput sequencing is a powerful technology to identify miRNAs. In this chapter, the methods used for construction of a small RNA library and high-throughput sequencing involving total RNA isolation, small RNA purification, adapter ligation, reverse transcription, PCR amplification, and Solexa sequencing are described. Bioinformatics and analysis of differential expression of miRNAs including primary disposal, miRNA identification, target prediction, and expression analysis are also discussed. These methodologies associated with identification and functional characterization of miRNAs may provide useful tools for readers to study miRNAs in plants in general and Medicago truncatula in particular.