Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. We have used chromatin immunoprecipitation and high throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome-wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.