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PREMISE The successful application of universal targeted sequencing markers, such as those developed for the Angiosperms353 probe set, within populations could reduce or eliminate the need for specific marker development, while retaining the benefits of full-gene sequences in population-level analyses. However, whether the Angiosperms353 markers provide sufficient variation within species to calculate demographic parameters is untested. METHODS Using herbarium specimens from a 50-year-old floristic survey in Texas, we sequenced 95 samples from 24 species using the Angiosperms353 probe set. Our data workflow calls variants within species and prepares data for population genetic analysis using standard metrics. In our case study, gene recovery was affected by genomic library concentration only at low concentrations and displayed limited phylogenetic bias. RESULTS We identified over 1000 segregating variants with zero missing data for 92% of species and demonstrate that Angiosperms353 markers contain sufficient variation to estimate pairwise nucleotide diversity (π)—typically between 0.002 and 0.010, with most variation found in flanking non-coding regions. In a subset of variants that were filtered to reduce linkage, we uncovered high heterozygosity in many species, suggesting that denser sampling within species should permit estimation of gene flow and population dynamics. DISCUSSION Angiosperms353 should benefit conservation genetic studies by providing universal repeatable markers, low missing data, and haplotype information, while permitting inclusion of decades-old herbarium specimens.