Abstract Human cancer cell lines and xenografts are valuable samples for whole-genome sequencing of human cancer. Tumors can be maintained by serial xenografting in athymic (nude) or severe combined immunodeficient (SCID) mice. Critical to next generation based sequencing is the relative proportions of human and mouse DNA. In the current study, we developed a molecular assay to quantify the relative contributions of human and mouse in mixed DNA samples. The assay was designed based on deletion/insertion variation between human and mouse genomes. The percentage of mouse DNA was calculated according to the relative peak heights of PCR products analyzed by capillary electrophoresis. Three markers from chromosomes 9 and 10 accurately predicted the mouse genome ratio and were combined into a multiplex PCR reaction. We used the assay to quantify the relative DNA amounts of 93 mouse xenografts used for a recently reported integrated genomic analysis of human pancreatic cancer. Of the 93 xenografts, the mean % of contaminating mouse DNA was 47%, ranging from 17% to 73%, with 43% of samples having greater than 50% mouse DNA. We then comprehensively compared the human and mouse genomes to identify 370 additional candidate gene loci demonstrating human-mouse length variation. With increasing use of whole genome sequencing of human cancers, this assay should be useful to monitor strategies to enrich human cancer cells from mixed human-mouse cell xenografts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2210.