Simple SummaryOur results demonstrated the dynamic changes in cow milk microbiota over the first two months of lactation, and the differences in microbial composition between primiparous cows and multiparous cows. The presence and fluctuation of some typically gut-associated microbes in milk indicated that the metabolic changes in dairy cows may influence the microbial structure of cow milk. These changes may potentially be correlated with milk components.AbstractThe present experiment was carried out to analyze the longitudinal changes in milk microorganisms. For this purpose, milk samples were collected from 12 healthy cows (n= 96; six primiparous cows and six multiparous cows) at eight different time points. The characteristics and variations in microbial composition were analyzed by 16S rRNA gene high-throughput sequencing. In the primiparous group, higher and more stable alpha diversity was observed in transitional and mature milk compared with the colostrum, with no significant difference in alpha diversity at each time point in the multiparous group. Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota were the most dominant phyla, andPseudomonas,UCG-005,Acinetobacter,Vibrio,Lactobacillus,Bacteroides,Serratia,Staphylococcus, andGlutamicibacterwere the most dominant genera in both primiparous and multiparous cow milk. Some typically gut-associated microbes, such asBacteroides,UCG-005, andRikenellaceae_RC9_gut_group, etc., were enriched in the two groups. Biomarker taxa with the day in time (DIM) were identified by a random forest algorithm, withStaphylococcusshowing the highest degree of interpretation, and the difference in milk microbiota between the two groups was mainly reflected in 0 d–15 d. Additionally, network analysis suggested that there were bacteria associated with the total protein content in milk. Collectively, our results disclosed the longitudinal changes in the milk microbiota of primiparous and multiparous cows, providing further evidence in dairy microbiology.