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Background: Total community rDNA was used to determine the diversity of bacteria and archaea from water, wet sediment and microbial mats samples of hot springs in the Eastern lowlands of Eritrea. The temperatures of the springs range from 49.5 °C to 100 °C while pH levels varied from 6.97 to 7.54. Akwar and Maiwooi have high carbonate levels. The springs near the seashore, Garbanabra and Gelti, are more saline with higher levels of sodium and chlorides. Elegedi, situated in the Alid volcanic area, has the highest temperature, iron and sulfate concentrations., Results: The five hot springs shared 901 of 4371 OTUs recovered while the three sample types (water, wet sediment and microbial mats) also shared 1429 OTUs. The Chao1 OTU estimate in water sample was significantly higher than the wet sediment and microbial mat samples. As indicated by NMDS, the community samples at genus level showed location specific clustering. Certain genera correlated with temperature, sodium, carbonate, iron, sulfate and ammonium levels in water. The abundant phyla included Proteobacteria (6.2-82.3%), Firmicutes (1.6-63.5%), Deinococcus-Thermus (0.0-19.2%), Planctomycetes (0.0-11.8%), Aquificae (0.0-9.9%), Chlorobi (0.0-22.3%) and Bacteroidetes (2.7-8.4%)., Conclusion: There were significant differences in microbial community structure within the five locations and sample types at OTU level. The occurence of Aquificae, Deinococcus-Thermus, some Cyanobacteria and Crenarchaeota were highly dependent on temperature. The Halobacterium, unclassified Thaumarchaeota, Actinobacteria and Cyanobacteria showed significant correlation with salinity occurring abundantly in Garbanabra and Gelti. Firmicutes and unclassified Rhodocylaceae were higher in the microbial mat samples, while Archaea were prominent in the wet sediment samples.