To find novel probes for duplex DNA, we prepared four types of triplexes containing a homopurine‐homopyrimidine 15‐mer duplex DNA, and examined their thermal stabilities (T _m values). The single strands used for triplex formation were a DNA 15‐mer having a defined C‐T mixed sequence, and its sugar‐modified analogs, namely 2′‐fluoro DNA, RNA, and 2′‐O‐methyl RNA. The 2′‐O‐methyl RNA and the RNA‐containing triplexes were similar in their enhanced stabilities at pH 6.1 and, amongst the four triplexes, the 2′‐O‐methyl was the most stable at pH 5.0. Furthermore, an experiment using a 34‐mer duplex DNA suggested that the 2′‐O‐methyl RNA‐triplex was destabilized, mostly as a result of the incorporation of a mismatched triplet, as compared to the DNA triplex counterpart. Thus, 2′‐O‐methyl RNA can serve as an effective probe for duplex DNA.