BACKGROUND: Precise genotyping of the intron 8 poly(TG) and poly(T) tracts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is of clinical relevance in CFTR pathology. The (TG)(m) locus influences the penetrance of the (T)(5) allele, which may be associated with male infertility by congenital bilateral absence of the vas deferens (CBAVD) or other CFTR-related disorders (CFTR-RD), in particular in the context of (TG)(12) and (TG)(13). Simple and accurate genotyping of both loci should thus be routinely offered in laboratories. METHODS: We designed a new single test method relying on multiplex allele-specific fluorescent PCR: (T)(5)-, (T)(7)-, and (T)(9)-specific primers, labeled with different fluorophores, in combination with a common primer. Each fluorescent PCR product was identified on a capillary sequencer by its fluorescence color, specific for (T)(n), and size, indicative of the (TG) length. We first validated the assay in 2 different laboratories on 52 DNA samples with already known genotypes. We then evaluated the method prospectively, compared with sequencing, on 62 samples from healthy individuals and 108 samples from patients with CBAVD or other CFTR-RDs. RESULTS: We observed a 100% match in both validation steps. Results found in CBAVD and CFTR-RD patients are in keeping with data in the literature. CONCLUSIONS: The assay proved to be simple, rapid, and accurate for single-test (TG)(m)(T)(n) genotyping and suited for analysis in clinical laboratories.