Background: The current NHSN guideline states that positive results from both blood cultures and non–culture-based testing (NCT) methodologies are to be used for central-line–associated bloodstream infection (CLABSI) surveillance determination. A positive NCT result in the absence of blood cultures or negative blood cultures in patients who meet CLABSI criteria is to be reported to NHSN. However, the reporting criteria for NCT changed starting January 1, 2020: If NCT is positive and the blood culture is negative 2 days before or 1 day after, the NCT result is not reported. If the NCT is positive with no blood culture within the 3-day window period, the NCT result is reported in patients who meet CLABSI criteria. We estimated the impact of the new NCT criteria on CLABSI numbers and rates compared to the previous definition. Methods: At our facility, the T2Candida Panel (T2), an NCT, was implemented for clinical use for the detection of early candidemia and invasive candidiasis. The T2 is a rapid molecular test performed directly on blood samples to detect DNA of 5 Candida spp: C. albicans/C. tropicalis, C. glabrata/C. krusei, and C. parapsilosis. In this retrospective study performed at an 877-bed teaching hospital in Detroit, we reviewed the impact of discordant T2 results (positive T2 with negative blood cultures) on CLABSI rates from January 1, 2017, to September 30, 2019, based on the current definition, and we applied the revised criteria to estimate the new CLABSI numbers and rates for the same period. Results: Of 343 positive T2 results, 202 (58.9%) were discordant and qualified for CLABSI determination during the study period. Of these, 109 (54%) met CLABSI criteria based on the current definition and 11 (5%) met CLABSI criteria using the new definition (proportional P < .001), resulting in an 89.9% reduction. The CLABSI rate per 1,000 central-line days, which includes discordant T2 results, based on the current and new NCT criteria, are listed in Table 1. Conclusions: In institutions that utilize NCT such as T2, application of the new 2020 NCT NHSN definition would significantly reduce the CLABSI number and have a significant impact on the CLABSI rates and standardized infection ratios (SIRs).