Genomic DNA (gDNA) libraries are prepared for next-generation sequencing (NGS) by fragmenting the gDNA and ligating adapter duplexes to the ends of the fragments. Interactions between gDNA fragments in a library can lead to inaccurate fragment size determinations. For example, heteroduplexes, caused by hybridization between partially homologous molecules, form if too many PCR cycles are performed on the library. We demonstrate here that the presence of heteroduplexes can alter the apparent size distribution of a gDNA library in an assay-specific manner. We also show that, despite their effects on an electropherogram, heteroduplexes do not actually alter the sequencing insert size or other NGS quality metrics.