This study reports the cryo-electron microscopy (cryo-EM) structural characterization of TDP-43 amyloid fibrils derived from the full-length protein. TDP-43 is a multifunctional protein involved in RNA-binding, phase separation, and alternative splicing. Abnormal aggregation of its C-terminal domain is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The research reveals that TDP-43 fibrils are polymorphic, containing three distinct amyloid structures. These structures differ in the number and orientation of protofilaments but share a similar fold containing an amyloid key motif, which is a unique arrangement of β-strands where strands interact through side chains rather than backbone hydrogen bonds. The study used cryo-EM to analyze TDP-43 fibrils formed in vitro from full-length TDP-43. The results show that the protein can form a polymorphic spectrum of fibril morphologies. The observed structures differ from previously described TDP-43 fibrils and provide insights into the structural diversity of amyloid fibrils derived from this protein. The fibrils were found to be polymorphic, with different morphologies varying in the number of protofilaments, their orientation, and the fold of the fibril protein. These variations may be influenced by incubation time and physical/chemical conditions during aggregation. The study identified two main fibril morphologies, Morphology-1 and Morphology-2, with Morphology-1 being more abundant. The structures were analyzed using cryo-EM, revealing three-dimensional maps with resolutions of 3.76 Å and 4.05 Å for Morphology-1a and 1b, respectively. The fibrils were found to be left-handed and polar, with different symmetries and numbers of protein stacks. The amyloid key motif was observed in all three structures, indicating a common structural element in TDP-43 amyloid fibrils. The study also identified aggregation-prone regions in TDP-43, which are not necessarily located within the fibril core but within the N-terminal domain and two RNA recognition motifs (RRMs). These regions are involved in the formation of intermolecular β-sheets and may drive the aggregation of TDP-43. The results suggest that the amyloid key motif is formed through a mechanism similar to the folding of a Greek key, with the polypeptide chain forming a U-shaped structure that allows the aggregation-prone regions to pair and fold back onto themselves. The study highlights the structural diversity of TDP-43 amyloid fibrils and their potential role in neurodegenerative diseases.This study reports the cryo-electron microscopy (cryo-EM) structural characterization of TDP-43 amyloid fibrils derived from the full-length protein. TDP-43 is a multifunctional protein involved in RNA-binding, phase separation, and alternative splicing. Abnormal aggregation of its C-terminal domain is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The research reveals that TDP-43 fibrils are polymorphic, containing three distinct amyloid structures. These structures differ in the number and orientation of protofilaments but share a similar fold containing an amyloid key motif, which is a unique arrangement of β-strands where strands interact through side chains rather than backbone hydrogen bonds. The study used cryo-EM to analyze TDP-43 fibrils formed in vitro from full-length TDP-43. The results show that the protein can form a polymorphic spectrum of fibril morphologies. The observed structures differ from previously described TDP-43 fibrils and provide insights into the structural diversity of amyloid fibrils derived from this protein. The fibrils were found to be polymorphic, with different morphologies varying in the number of protofilaments, their orientation, and the fold of the fibril protein. These variations may be influenced by incubation time and physical/chemical conditions during aggregation. The study identified two main fibril morphologies, Morphology-1 and Morphology-2, with Morphology-1 being more abundant. The structures were analyzed using cryo-EM, revealing three-dimensional maps with resolutions of 3.76 Å and 4.05 Å for Morphology-1a and 1b, respectively. The fibrils were found to be left-handed and polar, with different symmetries and numbers of protein stacks. The amyloid key motif was observed in all three structures, indicating a common structural element in TDP-43 amyloid fibrils. The study also identified aggregation-prone regions in TDP-43, which are not necessarily located within the fibril core but within the N-terminal domain and two RNA recognition motifs (RRMs). These regions are involved in the formation of intermolecular β-sheets and may drive the aggregation of TDP-43. The results suggest that the amyloid key motif is formed through a mechanism similar to the folding of a Greek key, with the polypeptide chain forming a U-shaped structure that allows the aggregation-prone regions to pair and fold back onto themselves. The study highlights the structural diversity of TDP-43 amyloid fibrils and their potential role in neurodegenerative diseases.