A hexanucleotide repeat expansion (HRE), (GGGGCC)ₙ, in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This study identifies a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology. The HRE forms DNA and RNA G-quadruplexes and promotes RNA•DNA hybrids (R-loops). Structural polymorphism causes repeat length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Nucleolin (NCL), an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. The results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies. The study provides a mechanistic model for repeat-associated neurodegenerative diseases. The C9orf72 HRE forms G-quadruplexes and R-loops, which impair RNA polymerase processivity and lead to abortive transcription. These structures sequester proteins that recognize their conformations, leading to nucleolar stress and neurodegeneration. The study also shows that the C9orf72 HRE causes nucleolar stress, leading to impaired rRNA processing and increased sensitivity to proteotoxic stress. The findings suggest that targeting these toxic nucleic acid conformations could be a potential intervention for the pathogenic cascade in ALS/FTD.A hexanucleotide repeat expansion (HRE), (GGGGCC)ₙ, in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This study identifies a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology. The HRE forms DNA and RNA G-quadruplexes and promotes RNA•DNA hybrids (R-loops). Structural polymorphism causes repeat length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Nucleolin (NCL), an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. The results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies. The study provides a mechanistic model for repeat-associated neurodegenerative diseases. The C9orf72 HRE forms G-quadruplexes and R-loops, which impair RNA polymerase processivity and lead to abortive transcription. These structures sequester proteins that recognize their conformations, leading to nucleolar stress and neurodegeneration. The study also shows that the C9orf72 HRE causes nucleolar stress, leading to impaired rRNA processing and increased sensitivity to proteotoxic stress. The findings suggest that targeting these toxic nucleic acid conformations could be a potential intervention for the pathogenic cascade in ALS/FTD.