The specificity and reliability of locked nucleic acid (LNA) substitution at the 3′ position of allele-specific PCR (AS-PCR) primers for SNP detection was investigated in direct comparison to DNA primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3′ LNA mismatch combinations were tested in triplicate with the plasmid target. LNA primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3′ LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3′ LNA primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3′ LNA primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications. Hum Mutat 22:79–85, 2003.© 2003 Wiley-Liss, Inc.