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Costa Rica is at the centre of the Mesoamerican biodiversity hotspot. Little is known about cyanobacteria from this region so far. Here, four isolates of the order Stigonematales (section V) were characterized in a polyphasic approach. All strains were isolated from geothermal sites and hot springs of Costa Rica. However, one of them, identified as Westiellopsis sp. Ar73, did not grow at more than 40°C. Based on its identical 16S rRNA to several previously isolated Westiellopsis sp. and Fischerella muscicola strains, a ubiquitous distribution throughout tropical and subtropical regions can be implied. In contrast, the isolates MV9, MV11 and RV14 grew well up to 50–55°C. Based on morphologic, ultrastructural, molecular and physiologic data, MV9, MV11 and RV14 were identified to belong to the genus Fischerella. Two distinct intergenic transcribed spacer (ITS) types, with or without tRNA genes, were detected for all Stigonematales analysed here, indicating ITS polymorphism as a characteristic feature of heterocystous cyanobacteria. In phylogenetic trees, these Fischerella spp. formed a new and distinct clade within the wider lineage of thermophilic Fischerella (Mastigocladus cf. laminosus), which might represent a geographic lineage. Thus, geographic isolation may be an underestimated aspect of microbial evolution. The strains presented here are suitable as new models to study this group of cyanobacteria.