The gut microbiota of fish plays a key role in many biological processes of their host. However, there is lack of understanding of gut microbial dynamics of the Nile tilapia (Oreochromis niloticus L. 1758) under various environmental conditions. This thesis examined the variations of gut microbiota across the gut region and sample types. The gut microbiota diversity varied significantly across the gut region (stomach, midgut and hindgut), but not between sample types (luminal content and mucosal). Sampling season and rearing environment (captive and wild-catch) showed a significant impact on microbial diversity. The alpha diversity displayed that the captive samples had higher diversity than the wild-catch Nile tilapia. Firmicutes and Fusobacteria were the most dominant phyla in the wild-catch samples, while Proteobacteria were dominant in the captive samples. The assessment of gut microbiota of Nile tilapia across lakes with altitude gradient displayed significant diversity variation between lakes, with low-altitude samples showing a higher alpha diversity. Firmicutes was dominant in Lake Awassa and Lake Chamo samples whereas Fusobacteriota was dominant in Lake Hashengie and Lake Tana samples. The ratio of Firmicutes to Bacteroidota was much higher in Lake Hashengie samples (altitude 2440 m) than in Lake Chamo (altitude 1235 m). The relative abundances of Actinobacteriota, Chloroflexi, Cyanobacteria, and Firmicutes were negatively correlated with altitude, while Fusobacteriota showed a positive association with altitude. Overall, this study showed that the gut microbiota of Nile tilapia is significantly affected by host factors such as gut regions, environmental factors such as season, rearing habitat, and altitude. Moreover, all samples shared a large core microbiota, which includes Proteobacteria, Firmicutes, and Fusobacteria. This study established the basis for future large-scale investigations of the gut microbiota of fishes in Ethiopian lakes. Keywords: Oreochromis niloticus, Nile tilapia, gut microbiome, amplicon-based metagenomic characterization, Ethiopian lakes, environmental factors