ABSTRACT: During the peri- and early post-partum period, the gastrointestinal tract (GIT) of the calf is colonised by a diverse microbiota. In the colon, this microbial community contributes to digestive activities, immune modulation, and overall calf health and productivity. However, the current knowledge of temporal microbial establishment in the neonatal hindgut is limited. This study focused on the ontogeny of colon microbiota establishment in Aberdeen Angus cross beef calves located across two farms, from birth through to post-weaning. Colon digesta samples were obtained from calves euthanised on days D0 (n = 7), D7 (n = 7), D14 (n = 5), D21 (n = 7), D28 (n = 5), and D96 (n = 7) of life. 16S rRNA amplicon sequencing was used to determine prokaryotic community composition. The alpha (α) and beta (β) diversity were assessed with age and farm included as fixed effects. Bacterial α-diversity increased significantly with age, showing changes in relative abundance between D7 and later stages, including D21, D28, and D96 (each p < 0.0001). PERMANOVA analysis indicated no significant difference in microbial composition between D14 and D21 (P = 0.22), suggesting a period during which compositional changes were reduced, reflecting a temporary phase of relative similarity in the colon microbiota. However, weaning altered the colon microbiota, as evidenced by the differences were observed between D28 and D96 (P < 0.001) suggested compositional shifts associated with weaning, accompanied by increased α-diversity. These observations will help to inform the design of the future, large scale studies aimed at understanding how early life microbial dynamics influence calf health, welfare, and future productive performance.