Methods to reliably assess the accuracy of genome sequence data are lacking. Currently completeness is only described qualitatively and mis-assemblies are overlooked. Here we present REAPR, a tool that precisely identifies errors in genome assemblies without the need for a reference sequence. We have validated REAPR on complete genomes or de novo assemblies from bacteria, malaria and Caenorhabditis elegans, and demonstrate that 86% and 82% of the human and mouse reference genomes are error-free, respectively. When applied to an ongoing genome project, REAPR provides corrected assembly statistics allowing the quantitative comparison of multiple assemblies. REAPR is available at http://www.sanger.ac.uk/resources/software/reapr/.