Podophyllotoxin is currently in high demand as the lead chemical precursor for the anti-cancer drugs etoposide and teniposide. We conducted a two-phase study: (i) screening of Juniperus and other related species and identified Juniperus virginiana as the species with the highest podophyllotoxin concentration and (ii) established the limits of variations of podophyllotoxin and essential oil content and composition in J. virginiana in the United States. In the second phase of the study, J. virginiana trees at 49 locations in Mississippi, Alabama, Tennessee, and North Dakota were sampled and analyzed for both podophyllotoxin and essential oil. Both the essential oil and podophyllotoxin had similar concentration ranges as percent of dry J. virginiana leaves. Hence the following qualitative grouping with respect to concentration of natural products in the leaves was assigned: very high (>0.3%), high (0.2–0.3%), medium (0.1–0.2%), and low (<0.1%). Based on the above qualitative grouping, the J. virginiana accession were divided into 10 different groups (chemotypes). The J. virginiana accessions also differed in essential oil composition, with wide variation of individual constituents. Based on the essential oil composition, the J. virginiana accessions were divided into the following groups (chemotypes): (1) safrole–limonene–linalool; (2) safrole–β-pinene–limonene–linalool; (3) β-pinene–limonene; (4) limonene; (5) limonene–linalool; (6) limonene–safrole; (7) limonene–safrole–β-pinene; (8) β-pinene–limonene–bornyl acetate; (9) β-pinene–limonene–linalool–bornyl acetate; and (10) myrcene–limonene chemotype. The essential oil of two of the J. virginiana chemotypes showed differential antioxidant activity. J. virginiana leaves, a by-product from the timber industry could be used as a sustainable source for both podophyllotoxin and essential oil. The availability of various chemotypes offers an opportunity for the development of cultivars for commercial production of podophyllotoxin and essential oil with specific compositional profile to meet the market requirements.