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Abstract Human mitochondrial DNA (mtDNA) genetic markers are abundant in sewage and highly human-specific, suggesting a great potential for the environmental application as human fecal pollution indicators. Limited data are available on the occurrence and co-occurrence of human mtDNA with fecal bacterial markers in surface waters, and how the abundance of these markers is influenced by rain events. A 1-year sampling study was conducted in a suburban watershed impacted by human sewage contamination to evaluate the performance of a human mtDNA-based marker along with the bacterial genetic markers for human-associated Bacteroidales (BacHum and HF183) and Escherichia coli. Additionally, the human mtDNA-based assay was correlated with rain events and other markers. The mtDNA marker was detected in 92% of samples (n = 140) with a mean concentration of 2.96 log10 copies/100 ml throughout the study period. Human mtDNA was detected with greater abundance than human-associated Bacteroidales that could be attributed to differences in the decay of these markers in the environment. The abundance of all markers was positively correlated with rain events, and human mtDNA abundance was significantly correlated with various bacterial markers. In general, these results should support future risk assessment for impacted watersheds, particularly those affected by human fecal pollution, by evaluating the performance of these markers during rain events.