Interception of potential invasive species at ports-of-entry is essential for effective biosecurity and biosurveillance programs. However, taxonomic assessment of the immature stages of most arthropods is challenging; characters for identification are often dependent on adult morphology and reproductive structures. This study aims to strengthen the identification of such specimens through DNA barcoding, with a focus on microlepidoptera. A sample of 241 primarily immature microlepidoptera specimens intercepted at U.S. ports-of-entry from 2007 to 2011 were selected for analysis. From this sample, 201 COI-5P sequences were generated and analyzed for concordance between morphology-based and DNA-based identifications. The retrospective analysis of the data over 10 years (2009 to 2019) using the Barcode of Life Data (BOLD) system demonstrates the importance of establishing and growing DNA barcode reference libraries for use in specimen identification. Additionally, analysis of specimen identification using public data (43.3% specimens identified) vs. non-public data (78.6% specimens identified) highlights the need to encourage researchers to make data publicly accessible. DNA barcoding surpassed morphological identification with 42.3% (public) and 66.7% (non-public) of the sampled specimens achieving a species-level identification, compared to 38.3% species-level identification by morphology. Whilst DNA barcoding was not able to identify all specimens in our dataset, its incorporation into border security programs as an adjunct to morphological identification can provide secondary lines of evidence and lower taxonomic resolution in many cases. Furthermore, with increased globalization, database records need to be clearly annotated for suspected specimen origin versus interception location.