The major milk whey proteins of the tammar wallaby (Macropus eugenii) have been identified over the total period of lactation using proteomic analysis techniques comprising two-dimensional electrophoresis, comparative image analysis, matrix assisted laser desorption ionisation mass spectrometry (MALDI MS), de novo peptide sequencing and cross species protein matching. Samples were collected at the periods coinciding with major milestones of immunological development in the developing marsupial and in the four phases of milk production, specifically, Days 0, 5 (Phase 1); 27, 68 (Phase 2A); 137, 174 (Phase 2B) and 250 (Phase 3). Major changes in the protein content of marsupial milk whey correlated with the changing needs of the pouch young for stages in growth and development. We have shown that the levels of milk whey proteins vary with the developmental stage of the young animal, with a high number of proteins detected in early and late milk compared with the middle phases of lactation. Over 41 proteins were confidently identified, of which most had known roles in immunological protection. Proteins providing immunological protection across the lactation period included transferrin, β2 microglobulin, haptoglobulin and a 78 kDa glucose regulated protein. Immunoglobulin IgJ linker chain and a known antimicrobial cathelicidin, were only detected for the first 100-137 days, after which time Complement B factor was found to be present (Phase 2B). The changes which correlated with development and growth in the pouch young were reflected by the presence of proteins such as an α-fetoprotein like protein and clusterin found in early milk (Phase 1-2A) and two unknown proteins which were apparent in very early mammary gland secretions. This is the first comprehensive proteomic study of the major whey proteins of a marsupial across the entire period of lactation and provides fundamental data on proteins secreted by the mammary gland during key stages of immunological development of the young animal.