Animal manures contain a large and diverse reservoir of antimicrobial resistance (AMR) genes that could potentially spillover into the general population through transfer of AMR to antibiotic-susceptible pathogens. The ability of poultry litter microbiota to transmit AMR was examined in this study. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 μg/mL), chloramphenicol (Cm; 25 μg/mL), streptomycin (Sm; 100 μg/mL), and tetracycline (Tc; 25 μg/mL). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genesaadA1andsul1, respectively, in the poultry litter community. AMR gene load was determined relative to total bacterial abundance using 16S rRNA qPCR. Poultry litter contained 108CFU/g, with Gram-negative enterics representing a minor population (<104CFU/g). There was high abundance of resistance to Sm (106to 107CFU/g) and Tc (106to 107CFU/g) and a sizeable antimicrobial-resistance gene load in regards to gene copies per bacterial genome (aadA1: 0.0001–0.0060 andsul1: 0.0355–0.2455). While plasmid transfer was observed fromEscherichia coliR100, as an F-plasmid donor control, to theSalmonellarecipient in vitro, no AMRSalmonellawere detected in a poultry litter microcosm with the inclusion ofE. coliR100. Confirmatory experiments showed that isolated poultry litter bacteria were not interfering with plasmid transfer in filter matings. As no R100 transfer was observed at 25 °C, conjugative plasmid pRSA was chosen for its high plasmid transfer frequency (10−4to 10−5) at 25 °C. WhileE. colistrain background influenced the persistence of pRSA in poultry litter, no plasmid transfer toSalmonellawas ever observed. Although poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low under conditions commonly used to assess plasmid conjugation.