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To clarify the changes in milk microbiota by lactation stage, i.e., d ays in milk (DIM) for Fresh of 0 to 21, Early (DIM of 22 to 80), Middle (DIM of 81 to 200), and Late (DIM 201 or more) lactation stages in dairy cows, we performed longitudinal (12 sampling at each stage, total 48 samples) and cross-sectional sampling (Fresh [n = 7], Early [n = 12], Middle [n = 11], and Late [n = 11] stages, total of 41 samples) to comprehensively analyze the microbiota in milk samples by 16S rRNA amplicon sequencing. Although the relative abundance of bacteria of the phylum Actinobacteria increased significantly in the Late lactation stage in the longitudinal study, no significant changes were observed in the cross-sectional study. While no changes were observed in the milk microbiota during the lactation stages, environmental factors appeared to have a comparatively larger impact than interindividual diversity on the composition of the milk microbiota. Furthermore, the findings illustrated the importance of selecting appropriate study designs to clarify changes in milk microbiota throughout the lactation period. The findings obtained in this study not only provide useful information for interpreting previous research results, but also provide knowledge that could be helpful when designing new studies.