AbstractIncorporation of noncanonical disulfide linkages into single‐domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T ms), reversibility of thermal unfolding, solubility, and antigen‐binding affinities of three types of sdAbs (VHH, VH, and VL domains). The Cys40‐Cys55 disulfide linkage was tolerated by 9/9 VHHs, 12/12 VHs, and 2/11 VLs tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40‐Cys55 disulfide linkage increased sdAb T m by an average of 10.0°C (range: 0–21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen‐binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.