AbstractPremiseThe use of DNA from herbarium specimens is an increasingly important source for evolutionary studies in plant biology, particularly in cases where species are rare or difficult to obtain. Here we compare the utility of DNA from herbarium tissues to their freezer‐stored DNA counterparts via the Hawaiian Plant DNA Library.MethodsPlants collected for the Hawaiian Plant DNA Library were simultaneously accessioned as herbarium specimens at the time of collection, from 1994–2019. Paired samples were sequenced using short‐read sequencing and assessed for chloroplast assembly and nuclear gene recovery.ResultsHerbarium specimen–derived DNA was statistically more fragmented than freezer‐stored DNA derived from fresh tissue, leading to poorer chloroplast assembly and overall lower coverage. The number of nuclear targets recovered varied mostly by total sequencing reads per library and age of specimen, but not by storage method (herbarium or long‐term freezer). Although there was evidence of DNA damage in the samples, there was no evidence that it was related to the length of time in storage, whether frozen or as herbarium specimens.DiscussionDNA extracted from herbarium tissues will continue to be invaluable, despite being highly fragmented and degraded. Rare floras would benefit from both traditional herbarium storage methods and extracted DNA freezer banks.