Rice paddy soil is a hotspot of antibiotic resistance genes (ARGs) due to the application of organic fertilizers. However, the temporal dynamics of ARGs in rice paddy soil and its flooded water during the growing season remain underexplored. In this study, a microcosm experiment was conducted to explore the ARG profiles in a long term (130 days) flooded two-phase manure-amended soil–water system. By using high-throughput quantitative PCR array, a total of 23–98 and 34–85 ARGs were detected in the soil and overlying water, respectively. Regression analysis exhibited significant negative correlations between ARG profile similarities and flooding duration, indicating that flooding significantly altered the resistome (P