Recent research suggests that the vast majority of the human genome codes for long processed transcripts (>200 nucleotides) that do not appear to code for proteins, referred to as long non-coding RNAs (lncRNAs). LncRNAs have been shown to be essential for a variety of biological processes, including muscle differentiation. Myogenesis is a tightly controlled process involving transcriptional regulation of thousands of genes, whose regulation by transcription factors and microRNAs has been well characterized. Here, we used transcriptome sequencing to explore the expression patterns of lncRNAs during skeletal muscle differentiation and identified more than 120 novel and known lncRNAs that are differentially expressed between myoblasts and myotubes. These include an intergenic lncRNA on chromosome 3, RMD-1, whose expression is induced 51-fold during myogenesis (q-value=0.0006). Expression analysis across twenty different human tissues showed almost exclusive expression of RMD-1 in skeletal muscle. We show that RMD-1 is conserved in mouse, and its expression is ~60-fold increased during myogenesis. Knockdown of RMD-1 in primary human myoblasts impaired myogenesis, based on morphological characteristics and expression of known markers of muscle differentiation. Using publicly available expression data, we found almost complete disruption of RMD-1 expression in the muscle of patients with Duchenne muscular dystrophy. These results suggest that RMD-1 is a highly potent, previously unrecognized regulator of myogenesis. Further characterization of the molecular mechanisms of RMD-1 action will lead to better understanding of the roles of this new regulatory factor in myogenesis, and will provide new insights into the developmental function of lncRNAs and their link to diseases.