Cannabis sativa is a wind-pollinated, predominantly dioecious, and outcrossing crop associated with high levels of genetic variability even within a single cultivar. As such, seed-grown crops are often constrained by variability issues, decreasing production efficiency and product consistency. F₁ hybrid seed technology offers great potential to address these limitations by generating genetically uniform populations from a cross of two inbred parental lines. In C. sativa, single-seed descent (SSD) is currently the most viable method to produce these homozygous parental lines necessary for F₁ hybrid seed production. This study exemplifies the potential of SSD coupled with chemically induced sex reversion to produce fully homozygous lines and its subsequent application in creating five F₁ hybrid accessions. Up to six rounds of SSD were performed in an 18-month period on 16 different lines, highlighting the speed of methodology. Inbreeding through XY males was most successful and offered the greatest advantages of the lines assessed. The F₁ hybrid lines were statistically more uniform than the inbred or original lines and more vigorous than the inbred lines, with F₁ lines increasing seed yield between 3.9% and 155% when compared to their midparents indicating the potential to exploit heterosis. Chemotype stability was achieved in some F₁ hybrid lines, showing that seed-grown cannabinoid crops would be possible in some contexts using F₁ hybrid methodology, paving the way for the validation of this breeding technique in field settings and highlighting a path toward commercial hybrid seed systems in C. sativa.

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