Cystic fibrosis (CF), a common genetic disorder, is due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Early diagnosis through newborn screening (NBS) benefits patients with CF and is now practiced throughout the United States, Australasia, and most western European countries. This guideline was developed with international consensus and describes comprehensively the laboratory tests for detecting CF risk among newborns as well as recommendations for follow-up care. The document focuses on the use of immunoreactive trypsinogen (IRT) assays and the detection of specific CFTR mutations with the IRT/deoxyribonucleic acid screening strategy. Special attention is given to CFTR mutations in geographically and ethnically diverse populations. A core panel of CFTR mutations is recommended with guidance included on its potential expansion. The intended target audience includes NBS laboratory and program personnel, regulatory agencies, CF center personnel, neonatologists, primary care providers, organizations responsible for networks of CF centers, and a variety of public health policy makers.