Protein thermostability has received growing attention in recent years. Little is known about the determinants of thermal resistance in individual protein families. However, it is known that the mechanism is family-dependent and not identical for all proteins. We present a multivariate statistical analysis to find the determinants of thermostability in one protein family, the serine hydroxymethyltransferase family. Based on principal component analysis, we identified three amino acid fragments as the potential determinants of thermostability. The correlation coefficients between all the putative fragments and the protein thermostability were significant according to multivariable linear regression. Within the fragments, four critical amino acid positions were identified, and they indicated the contributions of Leu, Val, Lys, Asp, Glu, and Phe to thermostability. Moreover, we analyzed the insertions/deletions of amino acids in the sequence, which showed that thermophilic SHMTs tend to insert or delete residues in the C-terminal domain rather than the N-terminal domain. Our study provided a promising approach to perform a preliminary search for the determinants of thermophilic proteins. It could be extended to other protein families to explore their own strategies for adapting to high temperature.