As part of the US National Park Service’s mandate to manage natural resources for present and future generations, we developed a long-term monitoring protocol for lakes and ponds in the Cape Cod National Seashore, Massachusetts, USA. These waters are a few 100s to > 10,000 years old and from 1-2 m to >20 m deep, oligotrophic to mesotrophic, acidic, and with low buffering capacity. Recreation, abutting development, and increases in groundwater withdrawals threaten water quality and aquatic life. We sampled littoral and deepwater benthos with dipnets and Ekman dredges, processed the samples through various-sized sieves, and developed data sets on shallow and deepwater aquatic communities. We used ordination and logistic regression to explore patterns in physical, chemical, and biological characteristics of the ponds and variables associated with species’ distributions. Few trends were evident. Ponds connected to the sea have higher pH and nutrients than isolated waters, and shallow, colored ponds differ from deep, clear lakes. Multi-year littoral sampling in two ponds shows distinct aquatic communities, but substantial between-year variation within ponds. We recommend deepwater sampling of Chaoboridae and Chironomidae as the most effective and efficient protocol for long-term monitoring of aquatic macroinvertebrates in freshwater ponds within the Seashore.