The present study was designed to evaluate the utility of environmental samples for convenient but accurate detection of avian influenza virus (AIV) in commercial poultry houses. First, environmental samples from AIV-negative commercial layer facilities were spiked with an H5N2 low pathogenic AIV and were evaluated for their effect on the detection of viral RNA immediately or after incubation at –20 C, 4 C, 22 C, or 37 C for 24, 48, or 72 hr. Second, Swiffer pads, drag swabs, and boot cover swabs were evaluated for their efficiency in collecting feces and water spiked with the H5N2 LPAIV under a condition simulated for a poultry facility floor. Third, environmental samples collected from commercial layer facilities that experienced an H5N2 highly pathogenic AIV outbreak in 2014–15 were evaluated for the effect of sampling locations on AIV detection. The half-life of AIV was comparable across all environmental samples but decreased with increasing temperatures. Additionally, sampling devices did not differ significantly in their ability to collect AIV-spiked environmental samples from a concrete floor for viral RNA detection. Some locations within a poultry house, such as cages, egg belts, house floor, manure belts, and manure pits, were better choices for sampling than other locations (feed trough, ventilation fan, and water trays) to detect AIV RNA after cleaning and disinfection. Samples representing cages, floor, and manure belts yielded significantly more PCR positives than the other environmental samples. In conclusion, environmental samples can be routinely collected from a poultry barn as noninvasive samples for monitoring AIV.