This study investigates the structural characterization of TDP-43 amyloid fibrils using cryo-electron microscopy (cryo-EM). TDP-43, a multifunctional protein involved in phase separation, RNA-binding, and alternative splicing, is known to aggregate in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The research reveals that TDP-43 fibrils are polymorphic, containing three distinct amyloid structures with different numbers and relative orientations of protofilaments. These structures share a similar fold with an amyloid key motif, which is distinct from the Greek key structure found in globular proteins. The observed fibril structures differ from previously described conformations and provide insights into the structural landscape of TDP-43 amyloid fibrils. The study also explores the aggregation-prone regions within the TDP-43 sequence, suggesting that the pairing of specific regions may drive the formation of amyloid fibrils. The findings contribute to a better understanding of the structural basis of TDP-43-related pathologies and offer potential implications for diagnosis and therapy.This study investigates the structural characterization of TDP-43 amyloid fibrils using cryo-electron microscopy (cryo-EM). TDP-43, a multifunctional protein involved in phase separation, RNA-binding, and alternative splicing, is known to aggregate in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The research reveals that TDP-43 fibrils are polymorphic, containing three distinct amyloid structures with different numbers and relative orientations of protofilaments. These structures share a similar fold with an amyloid key motif, which is distinct from the Greek key structure found in globular proteins. The observed fibril structures differ from previously described conformations and provide insights into the structural landscape of TDP-43 amyloid fibrils. The study also explores the aggregation-prone regions within the TDP-43 sequence, suggesting that the pairing of specific regions may drive the formation of amyloid fibrils. The findings contribute to a better understanding of the structural basis of TDP-43-related pathologies and offer potential implications for diagnosis and therapy.