Chroothece has been reported from a range of freshwater environments, including streams, shallow ponds, trickling water on cliffs and moist soils, mostly in Europe and North America. The identification of genera and species by morphology is difficult because of overlaps in critical characters. To help clarify diversity within the genus, samples from Spain and from other regions (UK and Guam, western Pacific) were compared. Ecological and morphological data from field and cultured material were correlated with molecular data (rbcL gene sequences) that differentiate two new species: Chroothece thermalis I. Chapuis, P. Sánchez, M. Aboal & O. Necchi Jr., sp. nov. in a thermal spring and Chroothece lobata M. Aboal, B. A. Whitton, I. Chapuis, P. Sánchez & O. Necchi Jr., sp. nov. in a semi-arid stream. The results suggest recognition of four species, C. thermalis, C. lobata, C. richteriana Hansgirg and C. rupestris Hansgirg, from Spain. Morphology and ecology are useful to help distinguish these species, but the genus needs further study for possible cryptic diversity.