Abstract The PRM/Alf inbred mice exhibit a huge intestinal lengthening. Since milk contains bioactive factors implied in numerous biological processes, one hypothesis is that PRM/Alf milk contains intestinotrophic factors contributing to this remarkable phenotype. A comparison between the milk from PRM/Alf and C57BL/6J (as a control) strains could be helpful in the identification of such factors, including proteins. However, a complete description of the mouse milk major protein fraction is still missing. Hence we adapted a reliable technique to separate and identify the major mouse milk proteins. This approach was achieved through the protein study of milk from C57BL/6J and PWK/Pas strains representative of two Mus musculus subspecies, M. m. domesticus and M. m. musculus respectively. C57BL/6J milk samples were first skimmed and fractionated by reverse phase-HPLC (RP-HPLC). The protein content of each chromatographic peak was analysed by SDS-PAGE and identified by mass spectrometry. This methodological approach allowed characterization of nine major mouse milk proteins: alpha(s1), beta, gamma, epsilon and kappa-caseins, Whey Acidic Protein, lactoferrin, Serum Albumin, Fatty Acid Binding Protein, as well as an alpha(s1)-casein isoform. Then, RP-HPLC patterns of C57BL/6J milk proteins were compared with those obtained starting from the milk of PWK/Pas females. This comparison revealed a protein polymorphism for the alpha(s1)-casein.