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Recognition of mismatches in DNA or RNA duplexes is important in a variety of molecular biology techniques, e.g., SNP analysis, real‐time PCR. Mismatch discrimination is evaluated from the difference of melting temperatures (T_m) between matched and single base mismatched duplexes. We examined these T_m differences for various chimeric probes where some nucleotides were modified with locked nucleic acids residues (LNA). Our goal was to develop general guidelines for design of probes that would improve mismatch discrimination relative to native DNA probes. Melting temperatures and thermodynamic stabilities were determined from UV melting experiments. Mismatch discrimination was found to be similar in both Na⁺ and Mg²⁺ buffers. Triplet of LNA modifications with the central base of the triplet at the mismatch site gives the highest difference of melting temperatures between perfectly matched and …