Antimicrobial resistance (AMR) threatens the health of humans and animals and has repeatedly been detected in wild animal species across the world. This cross-sectional study integrates whole-genome sequence data from Escherichia coli isolates with demonstrated phenotypic resistance that originated from a previous longitudinal wildlife study in southern Ontario, as well as phenotypically resistant E. coli water isolates previously collected as part of a public health surveillance program. The objective of this work was to assess for evidence of possible transmission of antimicrobial resistance determinants between wild meso-mammals, swine manure pits, and environmental sources on a broad scale in the Grand River watershed, and at a local scale-for the subset of samples collected on both swine farms and conservation areas in the previous wildlife study. Logistic regression models were used to assess potential associations between sampling source, location type (swine farm vs. conservation area), and the occurrence of select resistance genes and predicted plasmids. In total, 200 isolates from the following sources were included: water (n = 20), wildlife (n = 73), swine manure pit (n = 31), soil (n = 73), and dumpsters (n = 3). Several genes and plasmid incompatibility types were significantly more likely to be identified on swine farms compared to conservation areas. Conversely, internationally distributed sequence types (e.g., ST131), extended-spectrum beta-lactamase- and AmpC-producing E. coli were isolated in lower prevalences (<10%) and were almost exclusively identified in water sources, or in raccoon and soil isolates obtained from conservation areas. Differences in the odds of detecting resistance genes and predicted plasmids among various sources and location types suggest different primary sources for individual AMR determinants, but, broadly, our findings suggest that raccoons, skunks and opossums in this region may be exposed to AMR pollution via water and agricultural sources, as well as anthropogenic sources in conservation areas.