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Background    Phalaris  species ( Poaceae ) occupy diverse environments throughout all continents except Antarctica.  Phalaris arundinacea  is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for  Phalaris  barcoding as a DNA region with high nucleotide diversity for  Phalaris  species identification. Recent findings that  P. arundinacea  populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American  P. arundinacea  differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh)  Phalaris  specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic  Phalaris  specimens with known, extant  Phalaris  species.    Results  We obtained complete ITS1 and ITS2 sequences from 31  Phalaris  historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens .  Herbaria  Phalaris  specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%)  Phalaris  species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of  Phalaris  herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found.  Phalaris arundinacea  showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin.    Conclusions  While not as efficient as extant specimens to obtain DNA,  Phalaris  herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the  P. arundinacea  clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American  P. arundinacea . Our finding of misidentified  Phalaris  species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-021-03284-z.

Variability in ITS1 and ITS2 sequences of historic herbaria and extant (fresh) Phalaris species (Poaceae)