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The ∼407-myr-old Rhynie chert of Scotland contains exquisite body fossils of land plants, animals and microorganisms, which provide our earliest reasonably complete snapshot of a Phanerozoic terrestrial ecosystem. These fossils have been instrumental to our understanding of the 'greening of the land', a major transition in the history of the Earth–life system. Among the primary producers preserved in the chert are cyanobacteria, of which only a fraction have been formally described. Here, we report the occurrence of the colony-forming cyanobacterium Eoentophysalis in the Rhynie chert. To our knowledge, this represents the first bona fide record of Entophysalidaceae from any post-Cambrian fossil assemblage or any non-marine fossil assemblage of any age. The Rhynie Eoentophysalis appears remarkably similar in appearance both to modern marine and freshwater Entophysalis ssp. and to Eoentophysalis belcherensis , a shallow-marine fossil from the ∼2 Ga Belcher Group of Canada that is perhaps the oldest convincing cyanobacterium on record. Darkened cell envelopes in the Rhynie Eoentophysalis correspond well with both E. belcherensis and modern Entophysalis , whose cell envelopes often contain the photoprotective brown pigment scytonemin. The occurrence of Eoentophysalis in the Rhynie chert supports previous claims that the fossilisable traits of entophysalid cyanobacteria are evolutionarily static through geological time. These organisms may be such effective generalists that major changes in their environment – in this case, the transition to a fully non-marine habitat – have not imposed significant selection pressure on these traits. [ABSTRACT FROM AUTHOR]