Field-based techniques for genotyping and identification are facilitated by isothermal amplification and detection technology. This chapter focuses on developing a rapid and field-deployable method for early sex identification genotyping in plants. Leveraging the Y-chromosome-specific regions or gene segments, we designed and evaluated an alternative isothermal amplification assay through recombinase polymerase amplification (RPA) to detect male-specific DNA sequences and perform sex determination genotyping.