With over 280 spider (Araneae) species recorded within the State of Georgia, USA, the need for a well-documented natural history collection with a usable voucher system is critical to continually assess spider diversity and their future ecological impact in this region. Spider identification can be daunting for the inexperienced taxonomist; it is time consuming and sometimes requires destructive procedures. Previous works have successfully used an alternative method, DNA barcoding, to correctly identify spider species while preserving their morphology. This study set forth to create the core of a well-documented spider collection within Georgia Southern University’s Institute for Coastal Plain Science and use DNA barcoding as a diagnostic tool. We collected 334 spiders from varying locations within the Coastal Plain and optimized a DNA extraction protocol from spider legs. Cytochrome C Oxidase Subunit I (COI), the gene commonly used in DNA barcoding, and an additional nuclear gene Histone 3 type A (H3A) were amplified and sequenced from a total of 132 and 150 spiders, respectively. The COI dataset identified 16 families, 33 genera, and 36 different species, while H3A identified 15 families, 23 genera, and 15 species. In addition, based on these gene sequences, we generated phylogenetic inferences of the COI and, when possible, of a concatenated datase
