Sizing of DNA fragments is a routine analysis traditionally performed on agarose or polyacrylamide gels. Electrophoretic analysis is labor-intensive with only limited potential for automation. Recovery of DNA fragments from gels is cumbersome. We present data on automated, size-based separation of DNA fragments by ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) - DNA chromatography - on the WAVE DNA Fragment Analysis System with the DNASep cartridge. This system is suitable for accurate and rapid sizing of double-stranded (ds) DNA fragments from 50 to ca. 2000 base pairs (bp). Fluorescently labeled DNA fragments are compatible with the technology. Length-dependent separation of dsDNA fragments is sequence independent and retention times are highly reproducible. The resolving capabilities of DNA chromatography are illustrated by the analysis of multiple DNA size markers. Resolved dsDNA fragments are easily collected and are suitable for downstream applications such as sequencing and cloning. DNA chromatography under denaturing conditions with fluorescently labeled DNA fragments offers a means for the separation and purification of individual strands of dsDNA. Analysis of DNA fragments on the WAVE System is highly automated and requires minimal manual intervention. DNA chromatography offers a reliable and automated alternative to gel electrophoresis for the analysis of DNA fragments.