Breastmilk is a dynamic fluid which initial goal is to provide the most adapted nutrition to the neonate. Additional functions have been recently attributed to breastmilk, with the evidence of a specific microbiota and the presence of a variety of components of the immune system, such as cytokines and leukocytes. The composition of breastmilk varies through time, according to the health status of mother and child, and altogether contributes to future health of the infant. Obesity is a rising condition worldwide, that creates a state of systemic, chronic inflammation including leukocytosis. Here, we asked whether colostrum, the milk produced within the first 48 h post-partum, would contain a distinct leukocyte composition depending on the body mass index (BMI) of the mother. We applied a panel of 6 antibodies plus viability marker to the peripheral blood and colostrum obtained from obese (BMI > 30) and lean (BMI < 25) mothers to characterize 10 major leukocyte subpopulations using flow cytometry. While lymphoid cells were otherwise unaffected by their tissue of origin, the phenotypes of granulocyte and monocyte populations significantly contrasted between blood and colostrum, including variations in morphology and surface expression of CD45 and CD16. These differences recapitulated across groups, which suggests a generalized cell-specific phenotype alteration caused by trafficking to colostrum. The B lymphocyte compartment was significantly reduced in obese colostrum and these cells did not exhibit enhanced CD16 shedding in this tissue, unlike B lymphocytes from lean mothers’ colostrum. This is the first exhaustive characterization of major leukocyte subsets in obese mothers’ colostrum, and the first report of leukocyte subpopulations from Latin-American women’s colostrum. This pioneering study is a steppingstone to further investigate active immunity in human breastmilk.