We assessed the effect of closing live poultry markets in China on influenza A(H7N9) virus detection and viability. Intensive sampling was carried out before, during, and after a 2-week citywide market closure; the markets were cleaned and disinfected at the beginning of the closure period. Swab samples were collected at different sites within the markets and tested for H7N9 by real-time reverse transcription PCR and culture. During the closure, H7N9 viral RNA detection and isolation rates in retail markets decreased by 79% (95% CI 64%–88%) and 92% (95% CI 58%–98%), respectively. However, viable H7N9 virus could be cultured from wastewater samples collected up to 2 days after the market closure began. Our findings indicates that poultry workers and the general population are constantly exposed to H7N9 virus at these markets and that market closure and disinfection rapidly reduces the amount of viable virus.