Different demands on the muscles of mastication may influence their functional profile (size and distribution of muscle fibre types), which may change during growth and maturation, potentially influencing craniofacial growth. The aim of this study was to evaluate mRNA expression and cross-sectional area of masticatory muscle fibres compared with limb muscles in young and adult rats. Twenty-four rats were sacrificed at two different ages, namely 12 at 4 weeks (young) and 12 at 26 weeks (adult). The masseter, digastric, gastrocnemius and soleus muscles were dissected. Gene expression of myosin heavy-chain isoformsMyh7(MyHC-I),Myh2(MyHC-IIa),Myh4(MyHC-IIb) andMyh1(MyHC-IIx) in the muscles was measured using qRT-PCR RNA analysis, and immunofluorescence staining was performed to measure the cross-sectional area of different muscle fibre types. Different muscle types and ages were compared. Significant differences were found in the functional profile between masticatory and limb muscles. For the masticatory muscles, there was an increase inMyh4expression with age, and this change was more intense for the masseter muscles, which also presented an increase inMyh1expression, similarly to limb muscles. The fibre cross-sectional area of the masticatory muscles was generally smaller in young rats; however, this difference was less pronounced than in limb muscles.