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In this study, we sought to profile the abundances and drivers of antibiotic resistance genes in an urban river impacted by combined sewage overflow (CSO) events. Water samples were collected weekly during the summer for two years; then, quantitative PCR was applied to determine the abundance of resistance genes associated with tetracycline, quinolones, and β-lactam antibiotics. In addition to sampling a CSO-impacted site near the city center, we also sampled a less urban site ~12 km upstream with no proximal sewage inputs. The tetracycline genes <i>tetO</i> and <i>tetW</i> were rarely found upstream, but were common at the CSO-impacted site, suggesting that the primary source was untreated sewage. In contrast, <i>ampC</i> was detected in all samples indicating a more consistent and diffuse source. The two other genes, <i>qnrA</i> and <i>bla_TEM</i>, were present in only 40–50% of samples and showed more nuanced spatiotemporal patterns consistent with upstream agricultural inputs. The results of this study highlight the complex sources of ARGs in urban riverine ecosystems, and that interdisciplinary collaborations across diverse groups of stakeholders are necessary to combat the emerging threat of antibiotic resistance through anthropogenic pollution.