To protect environmental water from human fecal contamination, authorities must be able to unambiguously identify the source of the contamination. Current identification methods focus on tracking fecal bacteria associated with the human gut, but many of these bacterial indicators also thrive in the environment and in other mammalian hosts. Mitochondrial DNA could solve this problem by serving as a human-specific marker for fecal contamination. Here we show that the human mitochondrial hypervariable region II can function as a molecular fingerprint for human contamination in an urban watershed impacted by combined sewer overflows. We present high-throughput sequencing analysis of hypervariable region II for spatial resolution of the contaminated sites and assessment of the population diversity of the impacting regions. We propose that human mitochondrial DNA from public waste streams may serve as a tool for identifying waste sources definitively, analyzing population diversity, and conducting other anthropological investigations.