Human-specific Bacteroides HF183 (HS-HF183), human-specific E. faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., E coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% host-specificity. All the humans-specific markers showed > 97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10−5, 10−4 and 10−3 respectively. HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10−4 whilist EVs were detected up to dilution 10−5. The ability of the HS-HF183 marker to detect fresh sewage was 3 to 4 orders of magnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices were similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.