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Four new cyanobacterial strains isolated from biological desert crusts in Thar Desert, India were characterized using a polyphasic approach. The strains were designated to two mophotypes, but all strains exhibited identical 16S ribosomal RNA (rRNA) gene sequences. On the basis of 16S rRNA phylogenetic reconstruction, the strains belonged to the Oscillatoriales order and formed a coherent cluster in the phylogenetic tree, with more than 5% sequence divergence to the closest relative belonging to a species of Microcoleus Desmazieres ex Gomont. Our strains were different from the genus Microcoleus in phenotypic characters such as organization of thallus, trichome width, cell shape, gas vesicle, thylakoid arrangement and habitat. Although the strains shared some morphological similarities with members of Phormidiaceae, they differed in thylakoid pattern and 16S rRNA gene sequences. The type strain (PD2001/TDC17(T)) grew up to 45 degrees C with optimum growth between 30 and 35 degrees C. The sharp decrease in growth of that strain at 1% salinity indicates its sensitivity to salts. Phylogenetic, morphological, ultrastuctural and physiological analyses demonstrated that the investigated strains represented a novel cyanobacterial genus, for which the name Desertifilum tharense gen. et sp. nov. is proposed, with PD2001/TDC17(T) (= CCAP 1420/4(T) = BISR/CYANO/61(T)) as the type strain.