Testing immunoreactive trypsinogen (IRT) is the first step in cystic fibrosis (CF) newborn screening. While high IRT is associated with CF, some cases are missed. This survey aimed to find factors associated with missed CF cases due to IRT levels below program cutoffs. Twenty-nine states responded to a U.S-wide survey and 13 supplied program-related data for low IRT false screen negative cases (CFFN) and CF true screen positive cases (CFTP) for analysis. Rates of missed CF cases and odds ratios were derived for each factor in CFFNs, and two CFFN subgroups, IRT above ("high") and below ("low") the CFFN median (39 ng/mL) compared to CFTPs for this entire sample set. Factors associated with "high" CFFN subgroup were Black race, higher IRT cutoff, fixed IRT cutoff, genotypes without two known CF-causing variants, and meconium ileus. Factors associated with "low" CFFN subgroup were older age at specimen collection, Saturday birth, hotter season of newborn dried blood spot collection, maximum ≥ 3 days laboratories could be closed, preterm birth, and formula feeding newborns. Lowering IRT cutoffs may reduce "high" IRT CFFNs. Addressing hospital and laboratory factors (like training staff in collection of blood spots, using insulated containers during transport and reducing consecutive days screening laboratories are closed) may reduce "low" IRT CFFNs.