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Abstract Anaerobic gut fungi (AGF,Neocallimastigomycota) represent a phylum of zoospore-producing fungi inhabiting the gastrointestinal tracts of herbivores. Twenty mammalian-affiliated genera (M-AGF) and two tortoise-affiliated genera (T-AGF) have been described so far. Here, we report on three additional novel T-AGF isolates obtained from Texas and sulcata tortoises. Phylogenetic analysis using the D1-D2 regions of the large ribosomal RNA subunit (D1-D2 LSU), RNA polymerase II large subunit (RPB1), internal transcribed spacer-1 region (ITS1), and transcriptomics-enabled phylogenomic analysis clustered these strains into three distinct, deep-branching clades, closely related to previously described T-AGF genusTestudinimyces. All isolates displayed filamentous rhizoidal growth patterns and produced monoflagellated zoospores. Unique morphological characteristics included the production of elongated, thick, nucleated structures in GX isolates, the formation of thin hair-like projections on sporangial walls in SR isolates, and irregularly shaped sporangia in TM isolates. All strains grew optimally at 32-35 °C and showed distinct substrate utilization capacity (e.g., growth on pectin, chitin, galactose). LSU analyses revealed GX isolates as the first cultured representatives of tortoise-affiliated but previously uncultured lineage NY56, while SR and TM strains have not been encountered in prior culture-independent AGF surveys. We propose to accommodate these isolates in three new genera and species –Gopheromyces tardescens(GXA2),Gigasporangiomyces pilosus(SR0.6), andKelyphomyces adhaerens(TM0.3). Further, based on the ecological, physiological, and phylogenetic distinctions between T-AGF and M-AGF, we propose to establish a new family (Testudinimycetaceae) to accommodate the generaTestudinimyces, Gopheromyces,Gigasporangiomyces,andKelyphomyces, within a new order (Testudinimycetales), and amend the description ofNeocallimastigalesto circumscribe M-AGF genera only.