The aim of the study was to delineate and compare the proteomic maps of two muscles of dry-cured ham: the biceps femoris and the semimembranosus. For this purpose, we used two-dimensional electrophoresis on a subcellular muscle fraction: insoluble protein in low ionic strength buffer. After protein identification by MALDI–TOF mass spectrometry and bioinformatic analyses, we found differences in expression levels in the two muscles. Seventy-three proteins or fragments were differentially expressed: 43 were over-represented in semimembranosus and 30 in biceps femoris. Although the study was performed on the insoluble protein fraction in low strength ionic buffer, protein and fragment identifications by mass spectrometry showed that most of the proteins were involved in energy metabolism. The differences observed between the two muscles can be explained by the differences in salt and moisture content in the course of dry-cured ham processing.