Since the first H7N9 human case in Shanghai, February 19, 2013, the emerging avian-origin H7N9 influenza A virus has become an epizootic virus in China, posing a potential pandemic threat to public health. From April 2 to April 28, 2013, some 422 oral-pharyngeal and cloacal swabs were collected from birds and environmental surfaces at five live poultry markets (LPMs) and 13 backyard poultry farms (BPFs) across three cities, Wuxi, Suzhou, and Nanjing, in the Yangtze Delta region. In total 22 isolates were recovered, and six were subtyped as H7N9, nine as H9N2, four as H7N9/H9N2, and three unsubtyped influenza A viruses. Genomic sequences showed that the HA and NA genes of the H7N9 viruses were similar to those of the H7N9 human isolates, as well as other avian-origin H7N9 isolates in the region, but the PB1, PA, NP, and MP genes of the sequenced viruses were more diverse. Among the four H7N9/H9N2 mixed infections, three were from LPM, whereas the other one was from the ducks at one BPF, which were H7N9 negative in serologic analyses. A survey of the bird trading records of the LPMs and BPFs indicates that trading was a likely route for virus transmission across these regions. Our results suggested that better biosecurity and more effective vaccination should be implemented in backyard farms, in addition to biosecurity management in LPMs.