CD44 is a transmembrane protein that plays a role in cell–cell interactions and motility in a number of cell types. Cell–cell interactions are critical for myoblast differentiation and fusion but whether CD44 regulates myogenesis is unknown. Here, we show that CD44 plays a functional role in early myogenesis. Analyses of myofiber cross‐sectional area, after local injury in mouse tibialis anterior (TA) muscles, revealed that growth was transiently delayed in the absence of CD44. A muscle‐intrinsic role for CD44 is suggested as primary myoblasts from CD44^−/− mice displayed attenuated differentiation and subsequent myotube formation at early times in a differentiation‐inducing in vitro environment. Chemotaxis of CD44^−/− myoblasts toward hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was totally abrogated, although expression of their respective receptors did not appear to differ from wild‐type. Furthermore, motility of CD44^−/− myoblasts was decreased at early stages of differentiation as determined by time‐lapse microscopy. Wild‐type myoblasts contained two subpopulations of slow‐ and fast‐migrating cells, whereas CD44^−/− myoblasts were composed predominantly of the slower migrating subpopulation. Taken together, these data suggest that myoblast migration and differentiation are closely linked and CD44 is a key regulator. J. Cell. Physiol. 209: 314–321, 2006. © 2006 Wiley‐Liss, Inc.