Chromophoric dissolved organic matter (CDOM) is a large portion of the open ocean dissolved matter pool which contributes largely to ocean color. The composition and distribution of CDOM is essentially controlled by in-situ biological production, terrestrial inputs, photochemical degradation, and microbial consumption. Estuarine environments contain particularly diverse CDOM composition due to their large variety of inputs and shoreline land usage in addition to the mixing of freshwater and salt water. Developing a further understanding of CDOM variation and composition will help develop and improve satellite remote sensing algorithms, help us understand CDOM’s role in the global carbon, nitrogen, and oxygen cycles, and may help to prioritize in-situ sampling for water quality monitoring in areas of concern. The use of inherent optical properties combined with pH titration and chemical reduction with sodium borohydride (NaBH4), helps to probe the molecular composition of CDOM and its spatial variability. Detailed studies of CDOM from the Chesapeake Bay are limited with many studies only investigating the main channel of the Bay and neglecting the various tributaries. Also, there is a lack of studies which specifically probe the molecular composition of the CDOM samples. To address this, an in-depth analysis of the optical properties of CDOM and C18 extracted organic matter (C18-OM) from the Chesapeake Bay, focusing on various inputs, was performed. Chemical reduction with NaBH4 and pH titration were employed to probe the presence of specific functional groups and their contribution to overall optical properties, and how they vary between locations. Spectral slope (S300-700), E2:E3 absorption ratio, fluorescence intensity, and apparent quantum yield of fluorescence (AQY) were used to analyze 170 samples from various tributaries in the Chesapeake Bay. Overall, this study suggested 1) there may be multiple inputs of CDOM within the Chesapeake Bay 2) the Top of the Bay and central channel of the Bay are impacted by the heavy terrestrial input from the Susquehanna River 3) A lack of correlation between phytoplankton fluorescence and CDOM absorption suggest phytoplankton are not an immediate source of CDOM within the Chesapeake Bay and 4) removal of protein and phytoplankton fluorescence after sample filtration indicates these species must exist in aggregates >0.2 µm. Optical analysis combined with pH titration and NaBH4 reduction investigated the variation between 9 C18-OM extracts from various regions in the Chesapeake Bay and a humic material standard Suwannee River Fulvic Acid (SRFA). Additionally, this study investigated the validity of the Charge-Transfer (CT) model using the optical properties of model compounds. This study suggested 1) certain absorbing and emitting species are lost during C18 extraction but extracts are still representative of their CDOM 2) nearly identical optical responses to pH titration and NaBH4 reduction suggest similar chromophore content throughout the Chesapeake and 3) CT interactions leading to long wavelength absorption are more prevalent in Suwannee River Fulvic Acid (SRFA) than they are in the Chesapeake. To compare the molecular and optical properties of the Chesapeake Bay to other locales, these extracts were compared to extracts from the Delaware Bay (DEL), Equatorial Atlantic Ocean (EAO) and North Pacific Ocean (NPO) in addition to reference materials Suwannee River Fulvic Acid (SRFA), Pony Lake Fulvic Acid (PLFA), and Elliott Soil Humic Acid (ESHA). This study showed 1) composition of deprotonatable and reducible chromophores within the Chesapeake and Delaware Bays is nearly identical but different from the oceans 2) despite being estuaries and containing a mixture of fresh and ocean water, CDOM within both Bays looks terrestrially dominated 3) deep ocean extracts from the Atlantic and Pacific exhibit similar optical response to pH titration, NaBH4 reduction, and NaBH4 reduction combined with pH titration suggesting the similarity of deep ocean waters from both ocean basins.