The assembly of DNA barcode libraries is particularly relevant within species-rich natural communities for which accurate species identifications will enable detailed ecological forensic studies. In addition, well-resolved molecular phylogenies derived from these DNA barcode sequences have the potential to improve investigations of the mechanisms underlying community assembly and functional trait evolution. To date, no studies have effectively applied DNA barcodes sensu strictu in this manner. In this report, we demonstrate that a three-locus DNA barcode when applied to 296 species of woody trees, shrubs, and palms found within the 50-ha Forest Dynamics Plot on Barro Colorado Island (BCI), Panama, resulted in >98% correct identifications. These DNA barcode sequences are also used to reconstruct a robust community phylogeny employing a supermatrix method for 281 of the 296 plant species in the plot. The three-locus barcode data were sufficient to reliably reconstruct evolutionary relationships among the plant taxa in the plot that are congruent with the broadly accepted phylogeny of flowering plants (APG II). Earlier work on the phylogenetic structure of the BCI forest dynamics plot employing less resolved phylogenies reveals significant differences in evolutionary and ecological inferences compared with our data and suggests that unresolved community phylogenies may have increased type I and type II errors. These results illustrate how highly resolved phylogenies based on DNA barcode sequence data will enhance research focused on the interface between community ecology and evolution.