Cannabis sativa is one of the earliest plants to be domesticated for fiber, food and medicine. Seed from Cannabis grown for industrial purposes during the 18th through 20th centuries have escaped production and established feralized populations across the United States. To maximize the potential of feral Cannabis germplasm, determining the genetic structure and cannabinoid profile is crucial for selection and breeding of new compliant regionally adapted hemp cultivars. To resolve this, a collection of feral Cannabis, comprising 760 plants across twelve US states were sequenced using Genotyping-by-Sequencings (GBS), genotyped at the cannabinoid synthase (CBDAS) gene, and subject to gas chromatography-mass spectrometry (GC-MS) to assess cannabinoid profiles. Clustering analyses by ADMIXTURE and Principal Component Analysis (PCA) stratified the germplasm into five clusters (Mississippi-River, West North Central-b, West North Central-a, New York, and Indiana). The cannabinoid genotyping assay resolved the feral collections into Type I - B2/B2 (6%), Type II - B2/B1 (15%), and Type III - B1/B1 (78%). Total cannabinoid content ranged from 0.21 to 4.73%. The assessment of genetic diversity, population structure, and cannabinoid profile of the US feral Cannabis collection provides critical information and germplasm resources to develop new and improve existing hemp cultivars.