The increasing prevalence of antibiotic resistance genes (ARGs) in the environment is problematic due to the risk of horizontal gene transfer and development of antibiotic resistant pathogenic bacteria. Using a suite of monitoring tools, this study aimed to investigate the sources of ARGs in a rural river system in Nova Scotia, Canada. The monitoring program specifically focused on the relative contribution of ARGs from a single tertiarylevel wastewater treatment plant (WWTP) in comparison to contributions from the upgradient rural, sparsely developed, watershed. The overall gene concentration significantly (p < 0.05) increased downstream from the WWTP, suggesting that tertiarylevel treatment still contributes ARGs to the environment. As a general trend, ARG concentrations upstream were found to decrease as proximity to human-impacted areas decreased; however, many ARGs remained above detection limits in headwater river samples, which suggested their ubiquitous presence in this watershed in the absence of obvious pollution sources. Significant correlations with ARGs were found for HF183 human fecal marker, Escherichia coli, and some antibiotics, suggesting that these markers may be useful for prediction and understanding of ARG levels and sources in rural rivers.