This data release contains in situ winter cover crop biomass sampling data collected from fields located on the Delmarva Peninsula (Talbot, Queen Anne’s and Caroline County, Maryland, USA) and at the USDA-ARS Beltsville Agricultural Research Center (BARC), Beltsville, Maryland, USA. Samples were collected during the winter cover crop growing season, between November and April, from 2019 to 2023, and were analyzed for Nitrogen and Carbon content. The dataset also includes spectral reflectance data acquired from the European Space Agency’s Sentinel-2 Earth observing satellites on dates proximal to biomass collection, as well as fifteen spectral indices calculated from reflectance values. Each record includes information on planting date, location (Eastern Shore or BARC), species, season of sampling (fall, winter, spring), fertilization during the cover crop season, and weather variables from NLDAS-2 including growing degree days, frost degree days, photosynthetically active radiation, days below freezing, and longest consecutive frost since planting. Most samples also have fractional ground cover derived from red, green, blue (RGB) photography and measurements of canopy height. On-farm data geolocations are privacy protected under a research agreement with the Maryland Department of Agriculture, and are therefore not included in this Data Release. For access to geolocations, please contact the authors.