We previously generated a highly thermostable triple variant of Moloney murine leukemia virus reverse transcriptase, MM3 (E286R/E302K/L435R), by introducing positive charges by site-directed mutagenesis at positions that have been implicated in the interaction with template-primer (Yasukawa et al., (2010) J. Biotechnol., 150, 299-306). In this study, we attempted to further increase the thermostability of MM3. Twenty-nine mutations were newly designed, focusing on the number of surface charge, stabilization of hydrophobic core, and introduction of salt bridge. The corresponding 29 single variants were produced in Escherichia coli and characterized for activity and stability. Six mutations (A32V, L41D, L72R, I212R, L272E and W388R) were selected as the candidates for further stabilize MM3. Fifteen multiple variants were designed by combining two or more of the six mutations with the MM3 mutations, produced and characterized. The sextuple variant MM3.14 (A32V/L72R/E286R/E302K/W388R/L435R) exhibited higher thermostability than MM3.