We report a pair of simple PCR-RFLP assays based on two independently segregating nuclear genes which can differentiate North American native Phragmites australis subsp. 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 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 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 which also distinguishes 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.