**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** WITH INCREASED GLOBAL TRADE AND TRANSPORT, ANTHROPOGENIC MOVEMENT OF NON-INDIGENOUS PESTIFEROUS SPECIES IS BECOMING MORE COMMON. EFFECTIVE MANAGEMENT AND PREVENTION OF THESE INVASIONS RELIES ON ACCURATE SPECIES IDENTIFICATION AND PATHWAY ANALYSIS. HOWEVER, MANY OF THESE PESTS BELONG TO HIGHLY DIVERSE GROUPS OF CLOSELY-RELATED SPECIES THAT ARE DIFFICULT OR IMPOSSIBLE TO DELIMIT USING TRADITIONAL TAXONOMIC METHODS, AND TRADITIONALLY USED MOLECULAR MARKERS HAVE PROVEN INEFFECTIVE FOR BOTH IDENTIFICATION AND PATHWAY ANALYSIS PURPOSES. THIS PROJECT WILL USE CUTTING-EDGE PHYLOGENOMIC APPROACHES TO DESIGN DIAGNOSTIC TOOLS FOR SPECIES IDENTIFICATION AND PATHWAY ANALYSIS OF TWO HIGHLY PESTIFEROUS FAMILIES OF DIPTERA (TRUE FLIES), AND CREATE LABORATORY AND ANALYTICAL WORKFLOWS TO FACILITATE COST-EFFECTIVE AND RAPID USE OF SUCH DIAGNOSTIC TOOLS. BY FOCUSING ON PLANT AND ANIMAL PESTS IN THE ECONOMICALLY DAMAGING TEPHRITIDAE AND CALLIPHORIDAE, THE PROPOSED RESEARCH WILL DEMONSTRATE THE UTILITY, APPLICABILITY, AND UNIVERSALITY OF GENOMIC-BASED DIAGNOSTIC MARKERS AT MULTIPLE PHYLOGENETIC SCALES (I.E. DIPTERA-WIDE DOWN TO THE LEVEL OF SPECIES COMPLEXES AND POPULATIONS). THIS PROJECT IS AN INTERNATIONAL COLLABORATION INVOLVING TWO CO-PIS WITHIN USDA-ARS, AND SEVERAL COLLABORATING INVESTIGATORS FROM OTHER DOMESTIC AND INTERNATIONAL INSTITUTIONS, INCLUDING REGULATORY AGENCIES AND DIAGNOSTIC LABS. THE PROPOSED RESEARCH WILL PROVIDE CUTTING-EDGE TACTICAL PHYTOSANITARY TOOLS TO AID IN LONG TERM BIOSECURITY AND SUSTAINABILITY OF US AGRICULTURE, AND ADDRESSES MULTIPLE FARM BILL, NIFA, AND USDA-APHIS PRIORITY AREAS.