521 Background: Circulating tumor DNA (ctDNA) displays characteristics of an ideal serum biomarker. We sought to develop whole-exome sequencing of ctDNA to interrogate commonly mutated genes in renal cell carcinoma (RCC) for early tumor detection through a single blood sample. Methods: Patients with solid renal tumors and healthy controls provided 40 mL blood from which purified plasma cell-free DNA was prepared. A multiplex bar-coded polymerase chain reaction amplification using the Fluidigm Access Array was performed to prepare sequencing libraries for the Illumina HiSeq platform. Galaxy workflow was then utilized to identify mutations within the plasma cell-free DNA samples and results were compared to buffy coat sequencing containing control DNA for each individual’s sample. The following genes were queried: VHL, PBRM1, SETD2, BAP1, KDM5C, KIT, NFE2L2, MET, TP53, CDKN2A, FGFR3, PIK3CA, BRAF, and MUC4. Criteria for calling mutations included adequate frequency by overall count and percentage of reads, identification in all overlapping sequences and presence of buffy coat-derived control DNA for comparison with <0.5% containing the mutation. Results: Of the preoperative RCC patients, 20/30 (67%) had detectable somatic mutations compared to 2/48 (4.2%) controls. These included nonsynonymous, frameshift, stop-gain, and splice site mutations. Mutations were detected in RCC patients with both early and advanced stage disease, including a patient with a 1.1 x 0.7 x 0.5 cm tumor. Mutations were seen in all genes assayed. Conclusions: The majority of RCC patients of various stages and histology had ctDNA detected in a single preoperative blood sample. Comparatively, only two of the control patients sampled were ctDNA positive. Controls will be followed to identify the possible sources of ctDNA. Developing such non-invasive methods for RCC detection has the potential to enhance both diagnosis and surveillance of renal malignancy, even in the setting of small renal masses.