Anaerobic gut fungi (AGF) represent a basal fungal lineage (phylum Neocallimastigomycota) that resides in the rumen and alimentary tracts of herbivores. The AGF reproduce asexually, with a life cycle that involves flagellated zoospores released from zoosporangia followed by encystment, germination and the subsequent development of rhizomycelia. A fast and reliable approach for AGF spore collection is critical not only for developmental biology studies, but also for molecular biological (e.g. AMT-transformation and RNAi) approaches. Here, we developed and optimized a simple and reliable procedure for the collection of viable, competent, and developmentally synchronized AGF spores under strict anaerobic conditions. The approach involves growing AGF on agar medium in serum bottles under anaerobic conditions, and flooding the observed aerial growth to promote spore release from sporangia into the flooding suspension. The released spores are gently collected using a wide bore sterile needle. Process optimization resulted in the recovery of up to 7×10(9) spores per serum bottle. Further, the released spores exhibited synchronized development from flagellated spores to encysted spores and finally to germinating spores within 90min from the onset of flooding. At the germinating spore stage, the obtained spores were competent, and readily uptook small interfering RNA (siRNA) oligonucleotides. Finally, using multiple monocentric and polycentric AGF isolates, we demonstrate that AGF grown on agar surface could retain viability for up to 16weeks at 39°C, and hence this solid surface growth procedure represents a simple, cryopreservative- and freezing temperature-free approach for AGF storage.