The Solanaceae family is one of the largest and well-distributed plant families in the world. It contains species of agricultural and economical importance, such as Solanum tuberosum, Solanum melongena, Solanum lycopersicum, Nicotiana tabacum, and Capsicum annuum. In Puerto Rico, there are ≈46 species of Solanaceae of which six are endemic: Brunfelsia densifolia, Brunfelsia lactea, Brunfelsia portoricensis, Goetzea elegans, Solanum ensifolium, and Solanum woodburyi. Our objective was to use DNA barcoding to identify the Solanaceae species in Puerto Rico, including the endemics, and to assess the species relationships between them. To accomplish our objective, two chloroplast regions (psbA-trnH and matK) and a nuclear region [internal transcribed spacer (ITS)] were assessed. Pairwise distance and phylogenetic analysis demonstrate that DNA barcoding can be used to discriminate at the species level among these taxa in Puerto Rico. For all three markers, the genus that showed the highest pairwise distance between represented species was Solanum, whereas the genus that displayed the least was Capsicum. Phylogenetic trees of single and concatenated regions were generated from sequences obtained in this study and from data downloaded from the National Center for Biotechnology Information database. Our results show that this technique can be used to identify species with one, two, or three combinations of DNA barcode markers depending on the taxon. In addition, this is the first study to include the endemic species S. woodburyi in a molecular phylogenetic analysis, and it was found to have a close relationship with S. ensifolium, also endemic to Puerto Rico, and to Solanum bahamense from the Bahamas and Greater Antilles. Therefore, we suggest that S. woodburyi might be part of the Bahamense clade.