With the intensification of human activities, fresh water resources are increasingly being exposed to contamination from effluent disposal to land. Thus, there is a greater need to identify the sources and pathways of water contamination to enable the development of better mitigation strategies. To track discharges of domestic effluent into soil and groundwater, 10 synthetic double-stranded DNA (dsDNA)3 tracers were developed in this study. Laboratory column experiment and field groundwater and soil lysimeter studies were carried out spiking DNA with oxidation-pond domestic effluent. The selected DNA tracers were compared with a non-reactive bromide (Br) tracer with respect to their relative mass recoveries, speeds of travel and dispersions using the method of temporal moments. In intact stony soil and gravel aquifer media, the dsDNA tracers typically showed earlier breakthrough and less dispersion than the Br tracer, and underwent mass reduction. This suggests that the dsDNA tracers were predominantly transported through the network of larger pores or preferential flow paths. Effluent tracking experiments in soil and groundwater demonstrated that the dsDNA tracers were readily detectable in effluent-contaminated soil and groundwater using quantitative polymerase chain reaction. DNA tracer spiked in the effluent at quantities of 36 μg was detected in groundwater 37 m down-gradient at a concentration 3-orders of magnitude above the detection limit. It is anticipated it could be detected at far greater distances. Our findings suggest that synthetic dsDNA tracers are promising for tracking effluent discharges in soils and groundwater but further studies are needed to investigate DNA-effluent interaction and the impact of subsurface environmental conditions on DNA attenuation. With further validation, synthetic dsDNA tracers, especially when multiple DNA tracers are used concurrently, can be an effective new tool to track effluent discharge in soils and groundwater, providing spatial estimation on the presence or absence of contamination sources and pathways.