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Onsite molecular diagnostics can revolutionize fecal pollution source tracking. We aimed to validate a method for onsite qPCR assays with a miniature speaker-sized Q qPCR instrument and other portable equipment items. We showed that marker genes for total bacteria (16S) and <i>E. coli</i> (rodA) in 100 mL of river water measured with this method agreed within ±0.3 log₁₀ units with results obtained when using conventional laboratory equipment items. We then deployed the portable method in a mobile laboratory (‘lab in a van’) and quantified HF183 marker genes for human host associated <i>Bacteroides</i> in river water within 3 h of sampling. We also used the mobile laboratory to investigate urban river water and effluents from two storm drains and a retention pond and collected comprehensive microbial and physicochemical water quality data. We found significantly higher HF183 gene levels in the older storm drain compared to the river water (6.03 ± 0.04 vs. 4.23 ± 0.03 log₁₀ gene copies per 100 mL), and a principal component analysis revealed that storm drain effluent retention in a pond beneficially altered water characteristics, making them more like those of the receiving river. In conclusion, onsite qPCR assays can be performed with portable equipment items to quickly test water.