CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (AG) repeat in the gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the main pathogenic (AG) and the main nonpathogenic (AG) repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization . The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in supercoiled DNA reveals triplex H-DNA formation by the pathogenic repeat. Consistently, bioinformatic analysis of the S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (AG) motifs over (AG) motifs . Finally, the pathogenic, but not the non-pathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that CANVAS-causing (AG) repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.