In September 2007, an H7N3 highly pathogenic avian influenza outbreak (HPAI) occurred on a multiple-age broiler breeder operation near Regina Beach, Saskatchewan, Canada. Mortality was initially observed in a barn that housed 24-wk-old roosters, with later involvement of 32-wk-old breeders. All birds on the affected premises were destroyed, and surveillance of surrounding farms demonstrated no further spread. The use of water from a dugout pond during periods of high demand, and the proximity of the farm to Last Mountain Lake, the northern end of which is a bird sanctuary, implicated wild aquatic birds as a possible source of the virus. Of particular note, the H7-specific real-time reverse transcription polymerase chain reaction assay that was in use at the time did not detect the virus associated with this outbreak. A Canadian national influenza A virus survey of wild aquatic birds detected no H7 subtype viruses in 2005 and 2006; however, H7 subtype viruses were detected in the fall of 2007. Phylogenetic analysis of a number of these H7 isolates demonstrated an evolutionary relationship with each other, as well as with the H7N3 HPAI virus that was isolated from the Saskatchewan broiler breeder farm.