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The Cuatro Cienegas Basin (CCB) comprises several oligotrophic aquatic ecosystems limited by phosphorus. These aquatic systems are dominated by a high prokaryotic diversity, shaped by the stress of low nutrient supplies and interspecific competition. Although fungi constitute a diverse and important component of microbial diversity, the microfungal diversity in the CCB remains to be unveiled. With the aim to explore microfungal diversity and ecological patterns in this area, we present the first investigation analyzing cultivable taxa from sediment and water, as well as lignocellulolytic taxa obtained from incubated submerged plant debris, and wood panels in three contrasting freshwater systems in the CCB: Churince, Becerra and Pozas Rojas. We chose a culture-based approach to analyze sediment and water samples in order to obtain fungal cultures, providing opportunities for a posteriori studies, and the possibility of ex situ preservation of the diversity. We evaluated sequence data from the nuclear ribosomal internal transcribed spacer including the 5.8 rDNA region for 126 isolates, revealing 37 OTUs. These OTUs were phylogenetically affiliated to several genera in the fungal phyla: Zygomycota, Basidiomycota, and Ascomycota. We recorded two OTUs with saline affinity, agreeing with previous findings on the prokaryotic communities with ancestral marine resemblances. All the studied systems showed moderate diversity levels, however discrepancies among the diversity indexes were observed, due to the occurrence of abundant taxa in the samples. Our results indicated that lignocellulolytic microfungal communities are dominated by transient fungal taxa, as resident species were not recorded perhaps as a result of the long-term strong competition with the highly adapted prokaryotic community. Moreover, the obtained microfungal taxa occurred mostly on the resident plant debris, rather than submerged wood panels, perhaps as a result of the high adaptation to specific environmental conditions. In conclusion, the CCB possess a moderate taxonomical diversity compared to other arid environments, probably as a result of high selective pressures. Nonetheless, due to high spatial and temporal heterogeneity, the functional fungal diversity was considerable as predicted by the intermediate disturbance hypothesis. Decisively, the assessment of microfungal diversity freshwater systems is relevant, since this ecological group of microorganisms represents an important indicator of trophic complexity and biotic interactions among microbial communities, having important implications for understanding eukaryotic survival at the oligotrophic limit for life.