Microbial colonization in the gut during early life plays important roles in the host immunity development, metabolism, growth, and health. However, the knowledge on hindgut microbial colonization in dairy calves during the pre-weaned period is very limited. In this study, amplicon sequencing was used to characterize mucosal and digesta associated microbiota in the hindgut (cecum, colon and rectum) of newborn (at birth, n = 6), d 7 (n = 6), d 21 (n = 6), and d 42 (n = 6) Holstein bull calves. The comparison of the relative abundance of bacteria was performed with the nonparametric Kruskal-Wallis test. In total, 14 phyla were identified with Firmicutes, Bacteroidetes and Proteobacteria being the dominant phyla. At genus level, Lactobacillus and Bacteroides were the 2 predominant genera for both mucosa and digesta associated microbiota. The age effect was detected for both mucosa and digesta associated bacterial communities, while no regional effect was observed for them. Among the detected bacterial genera, the relative abundance of mucosa-attached Escherichia and Pseudomonas decreased significantly with the increase of age (P < 0.01), suggesting the initial higher prevalence of these potential pathogenic bacteria during first week of life but the prevalence lowered in healthy claves with the growth. The concentrations of total short-chain fatty acids (SCFAs), acetate, propionate, and butyrate were significantly higher at d 21 compared with d 7 (P < 0.01); however, there was no significant difference observed between d 21 and d 42 except for acetate concentration on cecum. The changing pattern of SCFA concentration was similar with the changes in the relative abundance of SCFA-producing bacterial genera such as mucosa and digesta associated Prevotella, Blautia, Ruminococcus, and mucosa-attached Fecalibacterium. This study provided the information on hindgut microbial composition and their metabolite, which may play an important role in the hindgut fermentation and health of dairy calves during pre-weaned period.