Abstract: Objective: 16S rDNA high-throughput sequencing was used to study the bacterial diversity of colostrum and normal milk of the three-breed crossbreds. Methods: The total bacterial DNA was extracted from the raw milk samples collected on the same day, the 16S rDNA of the bacteria was amplified by PCR, and the 16S rDNA library of the bacteria group was constructed by the purified amplified fragment. Miseq PE300 platform was used for sequencing and blast comparison. Results: There were 19 phyla, 292 genera and 437 species in colostrum group. 7 phyla, 203 bacterial genera and 330 bacterial species were obtained from the two groups of the normal milk group. The bacterial diversity of colostrum group was higher than that of the normal milk group. Chryseobacterium, Acinetobacter, and Pseudomonas were the dominant bacteria in the colostrum group, while Chryseobacterium, Lactococcus, and Enterococcus were the dominant bacteria in the normal group. Cluster analysis showed that there was no significant difference between the two groups. PCA and PLS-DA analysis showed that there were significant differences in bacterial flora structure between the two groups. Conclusion: The diversity of colostrum and normal milk of the three-breed crossbreds is rich, and the bacterial diversity in normal milk group is significantly higher than that in colostrum group.