Through the collection and sequencing of trace genomic evidence from environmental samples (e.g., water, air, and soil), environmental DNA (eDNA) metabarcoding can detect a range of vertebrates. Despite the dynamic characteristics of estuarine environments, which often hinder the persistence of genomic material, this project successfully employed metabarcoding to assess the distribution of vertebrates in the Chesapeake Bay. Primarily, the study evaluated the effects of using various eDNA sampling, laboratory, and post-hoc analysis techniques when investigating species presence and biodiversity of an area. This study also identified spatially-explicit fish communities along salinity gradients as described by a Generalized Additive Mixed Model (GAMM) and a Permutational Multivariate Analysis of Variance (PERMANOVA). Community compositions were similar to previous findings by traditional trawling and seining methods. This research supports the usefulness of eDNA metabarcoding to assess species presence across spatiotemporal extents, making it a promising tool for future biomonitoring efforts in the Chesapeake Bay.