Few studies have investigated heterogeneity of selection response in replicate lines subjected to equivalent selection. We developed four replicate lines of mice based on high levels of voluntary wheel running (high runner or HR lines) while also maintaining four nonselected control lines. This led to the unexpected discovery of the HR minimuscle (HRmini) phenotype, recognized by a 50% reduction in hindlimb muscle mass, which became fixed in 1 of the four HR selected lines. Here, we report genome-wide expression profiling describing transcriptome differences between HRnormal and HRmini medial gastrocnemius. Consistent with the known reduction of type IIB fibers in HRmini, Myh4 gene expression was -8.82-fold less (P = 0.0001) in HRmini, which was closely associated with differences in the "calcium signaling" canonical pathway, including structural genes (e.g., Mef2c, twofold greater in HRmini, P = 0.0003) and myogenic factors (e.g., Myog, 3.8-fold greater in HRmini, P = 0.0026) associated with slow-type myofibers. The gene that determines the HRmini phenotype is known to reside in a 2.6335-Mb interval on mouse chromosome 11 and 7 genes (Myh10, Chrnb1, Acadvl, Senp3, Gabarap, Eif5a, and Clec10a) from this region were differentially expressed. Verification by real-time PCR confirmed 1.5-fold greater (P < 0.05) expression of very long chain acyl-CoA dehydrogenase (Acadvl) in HRmini. Ten other genes associated with fatty acid metabolism were also upregulated in HRmini, suggesting differences in the ability to metabolize fatty acids in HRnormal and HRmini muscles. This work provides a resource for understanding differences in muscle phenotypes in populations exhibiting high running capacity.