In this study, the cell wall proteomes of 3 different regions of alfalfa stems were compared. These three regions correspond to three phases in the stem development. The sequential extraction of cell wall proteins with CaCl2, EGTA and LiCl-complemented buffers was combined with a gel-based proteome approach and multivariate analysis. Although the highest similarities were observed between the apical and intermediate stem regions, the three proteome patterns are characteristic for each region. In the basal stem region, proteins that bind carbohydrates and have proteolytic activity, as well as enzymes involved in glycan remobilization, accumulate. Beta-amylase and ferritin likewise accumulate more in the basal stem segment. Therefore remobilization of nutrients appears to be an important process in the oldest stem segment. The intermediate and the apical region are sites of cell wall polymer remodelling, as suggested by the high abundance of proteins involved in the remodelling of the cell wall such as xyloglucan endoglucosylase, polygalacturonase non-catalytic subunit or beta-galactosidase. The most striking change between the different stem parts is however the strong accumulation of a DUF642-conserved domain containing protein in the apical region of the stem which suggests a particular role of this protein during the early development of stem tissues.