The objective of this study was to evaluate the role of manure storage on antimicrobial and antimicrobial resistance (AMR) genes in swine manure and to quantify the transport of these constituents in runoff after land application of swine manure. To meet these objectives, a series of field rainfall simulation experiments and laboratory storage experiments were performed. In the rainfall simulation experiments, swine manure containing three different antimicrobials, chlortetracycline, tylosin, and bacitracin, was applied to land with three different methods: broadcast, surface incorporation, and injection. We determined that antimicrobials and AMR genes were detected in fresh swine manure, and that the concentration of both antimicrobials and the relative abundance of AMR genes decreased after storage of manure under anaerobic conditions. This indicates that on-site storage or holding of swine manure prior to land application will reduce the amount of antimicrobials and AMR genes present in the manure. Although storage decreased the levels of all AMR genes, the AMR genes associated with chlortetracycline (tetQ and tetX) were found to decrease more substantially compared with tylosin AMR genes, ermB and ermF. Results from the field rainfall simulation experiments indicated that AMR genes could be vertically transported in soil with infiltrating rainfall. The manure application strategy and timing of rainfall were found to be significant for predicting antimicrobial concentrations in runoff, with higher concentrations observed in runoff from plots receiving broadcast and incorporated manure compared with injected manure. In addition, decreasing concentrations were observed with increased time since manure application. To our knowledge, this is the first study to systematically investigate how different manure land application strategies affect antimicrobial and AMR gene levels in agricultural runoff and provide evidence that both manure storage and land application strategy can influence observed concentrations.