In mammals, separate muscles are typically specialized as a whole to provide distinct functional roles leading to well-recognized adaptations. This is exemplified in the lower limb by the slow, fatigue-resistant soleus, which provides a postural role vs. the fast, fatiguable tibialis anterior ( TA), which provides rapid movements. A unique characteristic of extraocular muscles (EOMs) is their compartmentalization into two distinct layers, the orbital layer (OL) and global layer (GL), presumably to subserve diverse functions within the same muscle. However, molecular evidence of this diversity has been limited. We used laser-capture microscopy coupled with microarray-based expression profiling to identify molecular differences between the OL and GL of rat EOMs. We found that 210 genes were differentially regulated between these layers at a twofold expression cutoff. Differences in genes related to metabolic pathways and related to structural elements of muscle and nerve formed the largest functional clusters. Layer-specific differential expression was validated at both mRNA and protein level for MYH3, MYH6, and ACTN3. The expected layer-specific differences among genes encoding vascular elements were not evident by profiling; morphometric analysis demonstrated that the differences exist, but at a magnitude below the cutoff level established by our statistical methods. Comparison of these results with previous results comparing whole EOMs and TA suggest evolutionary mechanisms may play a role in achieving functional distinctions between OL and GL.