The Momela soda lakes consist of seven small, hypersaline, alkaline lakes, situated in the East African rift valley at Arusha National Park, Tanzania. The lakes are fed by separate underground water sources with slightly varying mineral contents resulting in colour variation and supporting different kinds of prokaryotic and eukaryotic species. In this study, the diversity of Actinobacteria in surface water and sediments of five Lakes were investigated using culture-dependent and culture-independent molecular techniques. A total of 34 out of 112, and 13 out of 85, representatives of Actinobacteria isolates and clones, respectively, were selected for gene sequencing using the CD-HIT program. Analysis of their 16S rRNA gene sequences displayed the presence of species affiliated to 15 different genera, namely Mycobacterium, Rhodococcus, Microbacterium, Isoptericola, Dietzia, Leucobacter, Jonesia, Nesterenkonia, Micrococcus, Streptomyces, Hoyosella, Norcadiopsis, Cellulomonas, Bogoriella, and Agromyces. The results showed 5 and 12 putative new Actinobacteria isolates and clones, respectively. This is the first report of isolation of bacteria from the genus Mycobacterium from a soda lake globally, as well as the genera Hoyosella, Isoptericola, Jonesia, Micrococcus, Leucobacter and Agromyces from a soda lake in East Africa. Because Actinobacteria are known as a source of biotechnologically important compounds, the species revealed set a platform to search for novel bioactive compounds.