Background. Gestational Diabetes Mellitus (GDM) and obesity are threatening health conditions during pregnancy, as they affect the normal function of multiple systems, including neuro-hormonal networks, and adipose, liver, muscle and placenta. GDM and maternal obesity are main triggers to a vicious cycle of metabolic and cardiovascular diseases perpetuated trans-generationally. One of the first stages of this vicious cycle occurs during early lactation as the infant feeds of “disbalanced” breastmilk microbiota and macromolecule level. Despite the importance of breastmilk microbiota on newborn development, few studies have characterized breastmilk microbiota in association with obesity and GDM. Maternal obesity decreases the diversity of breastmilk microbiota, with increased proportions Staphylococcus compared to Bifidobacterium and Bacteroides. However, the extent to which GDM together with maternal obesity affect breastmilk microbiota is unknown. Here, we applied 16S-rRNA high-throughput sequencing to characterize colostrum microbiota of 43 mothers with exclusive GDM and obesity in order to address the impact of GDM/obesity on breastmilk microbiota.Results. We identified a total of 1,496 amplicon sequence variants (ASVs), being Proteobacteria and Firmicutes the dominant phyla. We found Staphylococcus, Corynebacterium-1, Anaerococcus and Prevotella overrepresented in samples of women with obesity and women with GDM. Population diversity indicators, such as Shannon/Faith phylogenetic index and UniFrac/robust Aitchison distances show distinct microbial composition for GDM (female-newborn subgroup) and obesity (male-newborn subgroup) compared to controls. Finally, Differential abundance analysis showed that Rhodobacteraceae was distinct for GDM and five families (Bdellovibrionaceae, Halomonadaceae, Shewanallaceae, Saccharimonadales and Vibrionaceae) were distinct for obesity groups.Conclusions. Our study indicates that GDM and obesity are related to a higher microbial diversity. Preliminary functional analysis suggests that this could be attributed to overrepresentation of amino acid and carbohydrate metabolism bacterial pathways. However, there is a strong influence of the infant´s gender and the utilization of antibiotics intrapartum. To our knowledge, this study represents the first characterization of the taxonomical changes of colostrum breastmilk in mothers with GDM. We think this study contributes to future design of functional metagenomic studies aiming to understand molecular mechanisms by which breastmilk from mothers with GDM/obesity affect the development and future adult physiology of their suckling infants.