In soybeans, eighteen members constitute the serine hydroxymethyltransferase (GmSHMT) gene family, of which the cytosolic-targeted GmSHMT08c member has been reported to mediate resistance to soybean cyst nematode (SCN). This work presents a comprehensive study of the SHMT gene family members, including synteny, phylogeny, subcellular localizations, haplotypes, protein homology modeling, mutational, and expression analyses. Phylogenetic analysis showed that SHMT genes are divided into four classes reflecting their subcellular distribution (cytosol, nucleus, mitochondrion, and chloroplast). Subcellular localization of selected GmSHMT members supports their in-silico predictions and phylogenetic distribution. Expression and functional analyses showed that GmSHMT genes display many overlapping, but some divergent responses during SCN infection. Furthermore, mutational analysis reveals that all isolated EMS mutants that lose their resistance to SCN carry missense and nonsense mutations at the GmSHMT08c, but none of the Gmshmt08c mutants carried mutations in the other GmSHMT genes. Haplotype clustering analysis using the whole genome resequencing data from a collection of 106 diverse soybean germplams (15X) was performed to identify allelic variants and haplotypes within the GmSHMT gene family. Interestingly, only the cytosolic-localized GmSHMT08c presented SNP clusters that were associated with SCN resistance, supporting our mutational analysis. Although eight GmSHMT members respond to the nematode infestation, functional and mutational analysis has shown the absence of functional redundancy in resistance to SCN. Structural analysis and protein homology modeling showed the presence of spontaneous mutations at important residues within the GmSHMT proteins, suggesting the presence of altered enzyme activities based on substrate affinities. Due to the accumulation of mutations during the evolution of the soybean genome, the other GmSHMT members have undergone neofunctionalization and subfunctionalization events.