We report a pair of simple polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assays based on two independently segregating nuclear genes that can differentiate the North American native [Phragmites australis (Cav.) Trin. ex Steud. ssp. americanus Saltonst., P.M. Peterson & Soreng] from other lineages. Because nuclear markers are inherited biparentally, researchers can also use them to screen for F1 hybrids between P. australis ssp. americanus and the other lineages. We show that a previously described assay based on an indel in the nuclear gene NRT2 consistently identifies a wide range of P. australis ssp. americanus haplotypes and distinguishes them from the Gulf Coast type [Phragmites australis (Cav.) Trin. ex Steud. ssp. berlandieri (E. Fourn.) Saltonst. & Hauber]. We also demonstrate a new PCR-RFLP assay for a previously described diagnostic single-nucleotide polymorphism adjacent to the PaGT4 microsatellite marker that also distinguishes P. australis ssp. americanus from the other lineages. In addition, we report the first case of Asian haplotype AS identified in North America and make recommendations for its detection. Our findings expand the tools available to those monitoring for invasion by introduced Phragmites in North America.