The development and first application of the sterile insect (SIT) technique was instrumental at eliminating screwworm, Cochliomyia hominivorax, from North and Central America. A permanent control zone was established along the Panama- Colombia border in 2006, with constant sterile releases preventing incursion since its implementation. However, screwworm cases in Panama began increasing in 2022 and have since spread west into Nicaragua. The direct cause of the increase in cases is unknown as mass-reared screwworm flies consistently met or exceeded quality control parameters. To evaluate possible causes for the outbreak, wild lines sourced from outbreak cases were reared in parallel with the production strain and quality parameters were evaluated. Behavior differences were observed with updated mating competition assays and the adaptation of flight mills and locomotor activity monitors to screwworm. Few significant differences were observed in quality control metrics and no physical measurements were different between the lines. Competitive mating assays revealed that the wild line mated nearly twice as many females as the production strain and did not perform differently throughout the day. In contrast, the production males mated more in the early morning, but mating success decreased later in the day. These nuanced differences in mating behavior were not captured by standard quality measurements, highlighting the necessity for further refinement and development of novel quality control metrics used for evaluating efficacy of new strains that are in development.