Objectives: In 2019, the Council of State and Territorial Epidemiologists ratified a multitiered standardized surveillance case definition (SSCD) for neonatal abstinence syndrome (NAS) to minimize variability in definitions across states. This evaluation of the tier 1 NAS SSCD aimed to identify common challenges and opportunities for enhancement to support consistent implementation of the definition. Methods: This mixed-methods analysis consisted of 3 virtual focus groups in March 2021 with site principal investigators, medical record abstractors, and data analysts (1 focus group each) from 4 jurisdictions piloting the tier 1 NAS SSCD. We analyzed focus group transcripts to create a codebook. We collected written reports in February 2022 from the 4 jurisdictions, conducted thematic analysis of focus group transcripts and written reports to identify themes, and collected surveillance data on infants identified with NAS born from January 2020 through December 2021 from the pilot sites. We analyzed surveillance data to further inform identified themes. We examined agreement among tier 1 classifications assigned independently by each pilot site and the Centers for Disease Control and Prevention to cases of NAS. Results: Three major themes emerged in the data: challenges abstracting data on withdrawal signs from the medical record, difficulty determining the time frame of prenatal substance exposure, and challenges assigning case classifications. In a comparison of tier 1 classifications assigned by the Centers for Disease Control and Prevention and the sites, 82.1% of cases in the dataset were concordant. Conclusions: We identified several opportunities to modify the SSCD to promote consistency and ease implementation across jurisdictions. Promoting consistent implementation supports comparability of NAS incidence estimates across jurisdictions, evaluation of prevention efforts, and allocation of resources to support families.