Environmental DNA (eDNA) sampling has become a widespread approach for detecting aquatic animals with high potential for improving conservation biology. However, little research has been done to determine the size of particles targeted by eDNA surveys. In this study, we conduct particle distribution analysis of eDNA from a captive Brook Trout (Salvelinus fontinalis) in a flow-through mesocosm. Our data suggest that 1.2–10-μm particles are the most common size of eDNA from Brook Trout, which is consistent with our hypotheses that eDNA in the environment is comprised of loosely aggregated smaller particles, resulting in high inter-sample heterogeneity. These findings are similar to those of a study on Common Carp (Cyprinus carpio) in lentic systems.