High throughput method to genotype plants

Methods are provided for high throughput genotyping of plants, utilizing at least three primers, one primer recognizing a polymorphic sequence of a first species of a plant genus, a second primer recognizing a second polymorphic sequence of a second species, and a third primer that recognizes sequences of both the first and second species and producing a measurable signal when amplifying a plant DNA-containing sample. Additional primers recognizing additional species may also be employed. The method may be repeated for multiple sequences each diagnosing a species or hybrid, and results analyzed using data from multiple assays to improve the statistical robustness of genotyping results. Controls are provided in which the primer target sequences are introduced into and extracted from bacteria and the measurable signal used as a control. The methods are particularly useful for genotyping a population of plants, especially where weed species and/or hybrids are present.