Abstract The freshwater thermal springs of Zerka Ma'in, located in Jordan in the mountains of Moab east of the Dead Sea, are densely inhabited by cyanobacteria up to the highest temperature of 63°C. We have investigated the cyanobacterial diversity of these springs and their outflow channels by microscopic examination, culture-dependent and culture-independent phylogenetic analysis, and by physiological studies of selected isolates of special interest. Both unicellular and filamentous types of cyanobacteria are present, and we identified morphological types such as Thermosynechococcus, Chroogloeocystis, Fischerella (Mastigocladus), Scytonema (occurring as large masses at lower temperatures), and others. Although morphologically similar cyanobacteria have been identified in hot springs world-wide, the Zerka Ma’in strains were phylogenetically distinct based on 16S rRNA gene sequence analysis. Considerable diversity was detected also in the gene sequences of nifH (nitrogenase reductase), encoding one of the key enzymes involved in nitrogen fixation. Nitrogen fixation in a Mastigocladus isolate obtained from the springs was investigated in further depth. The heterocystous strain could fix nitrogen (as assayed by acetylene reduction) at tem peratures up to 53°C.