The use of molecular methods, such as the detection of environmental deoxyribonucleic acid (eDNA), have become an increasingly popular tool in surveillance programs that monitor for the presence of invasive species in aquatic systems. One early application of these methods in aquatic systems was surveillance for DNA of Asian carps (specifically bighead carp Hypophthalmichthys nobilis and silver carp H. molitrix) in water samples taken from the Chicago Area Waterway System. The ability to identify DNA of a species in an environmental sample presents a potentially powerful tool because these sensitive analyses can presumably detect the presence of DNA in water even when the species is not abundant or are difficult to catch or monitor with traditional gear. Prior to research presented in this report, an initial eDNA surveillance effort was completed in selected locations in the Upper Mississippi and St. Croix Rivers in 2011 after the capture of a bighead carp in the St. Croix River near Prescott, WI. Data presented in this report were developed to duplicate the 2011 monitoring results from the Upper Mississippi and St. Croix Rivers and to provide critical insight into the technique to inform future work in these locations. We specifically sought to understand the potential confounding effects of other pathways of eDNA movement (e.g., fish-eating birds, watercraft) on the variation in background DNA by collecting water samples from (1) sites within the St. Croix River and the upper Mississippi River where the DNA of silver carp was previously detected, (2) sites considered to be free of Asian carp, and (3) a site known to have a large population of Asian carp. We also sought to establish a baseline Asian carp eDNA signature to which future eDNA sampling efforts could be compared. All samples taken as part of this effort were processed using conventional polymerase chain reaction (PCR) according to procedures outlined in the U.S. Army Corps of Engineers Quality Assurance Project Plan with minor deviations designed to enhance the rigor of our data. Presence of DNA in PCR-positive samples was confirmed by Sanger sequencing (forward and reverse) and sequences were considered positive only if sequences (forward and reverse) of ≥150 base pairs had a match of ≥95% to those of published sequences for bighead carp or silver carp. The DNA of bighead carp and silver carp was not detected in environmental samples collected above and below St. Croix Falls Dam on the St. Croix River, above and below the Coon Rapids Dam and below Lock and Dam 1 on the Upper Mississippi River, and from two negative control lakes, Square Lake and Lake Riley. The DNA of silver carp was detected in environmental samples collected below Lock and Dam 19 at Keokuk, Iowa, a reach of the river with high silver carp abundance. The portion (68%) of environmental samples taken below Lock and Dam 19 that were determined to contain the DNA of silver carp was similar to that reported in the scientific literature for other abundant species. The DNA of bighead carp, however, was not detected in environmental samples collected below Lock and Dam 19, a reach of the river known to have bighead carp. Previous reported detections of the DNA of silver carp in samples collected in 2011 were not replicated in this study. Additional analyses are planned for the DNA extracted from the samples collected in 2012. Those analyses may provide additional information regarding the lack of amplification of bighead carp DNA and the lengths of the sequences of silver carp DNA present in samples taken below Lock and Dam 19. These additional analyses may help inform the use of eDNA monitoring in large, complex systems like the Mississippi River.