Specific conductance (SC), soil volumetric water content (VWC), and discharge were monitored on a subsurface agricultural drain for a 2-yr period (2007–2008) to differentiate preferential flow paths from matrix flow paths. A major observation from the 2-yr period was the fast SC decrease after relatively small rainfall events, often <5 mm. A total of 25 paired rainfall–SC events were classified, with an average preferential flow onset time (from the event start) after 1.7 h and maximum preferential flow after 2.4 h. A specific conductance end-member mixing analysis (SC-EMMA) was used to determine the volume of water that infiltrated through preferential flow pathways. The SC-EMMA was used for 20 of the 25 paired rainfall–SC events; of the 20 classified events, the maximum preferential flow ranged from 11 to 75% of the total subsurface drain flow, with a mean maximum preferential flow of 31%. Overall, SC-EMMA illustrated that a significant portion of the subsurface drain discharge can be attributed to preferential flow, mainly through macropores or other largely open preferential flow pathways. The other primary mechanism, antecedent moisture conditions shifts, could only be shown for four of the 25 classified events. Specific conductance as a tracer of preferential flow was shown to be an effective tool for distinguishing preferential flow to subsurface drains. Even during relatively dry periods, the SC had a substantial decrease shortly after a rainfall event contrary to the conventional idea that macropore flow starts only after all the smaller pores are saturated and surface ponding begins to occur.