Glutathione S-transferases (GSTs) are essential drug-metabolizing enzymes, involved in conjugation of various endogenous and exogenous substrates. Cynomolgus macaque is an important primate species in drug metabolism studies; however, cynomolgus GSTs have not been fully characterized. In this study the cDNAs of 12 GSTs (GSTA3-A5, GSTK1, GSTM2-M4, GSTO2, GSTP1, GSTS1, and GSTT1/2) were isolated from cynomolgus macaque and rhesus macaque liver. Cynomolgus GSTM1 cDNA was not amplified and only an aberrantly spliced GSTM1 transcript was isolated from rhesus macaque. Amino acid sequences of these 12 GSTs shared high sequence identities (93–98%) and were clustered into the same clades as the human orthologs in the phylogenetic tree. The 12 GSTs had exon–intron structures similar to the human orthologs, and exhibited distinct tissue expression patterns. GSTA3, GSTA5, and GSTM3/O2 were expressed predominantly in adrenal gland, jejunum, and testis, respectively, whereas the other GSTs showed universal expression patterns in the 10 tissues analyzed. Comparison of expression levels showed that GSTA1, GSTK1, GSTA3, and GSTM3 were most abundantly expressed in liver/jejunum, kidney, adrenal gland, and testis, respectively. Metabolic assays of proteins expressed heterologously in Escherichia coli, showed that all 12 GSTs and 5 previously identified GSTs, GSTA1/2, GSTM5, GSTO1, and GSTZ1, catalyzed the conjugation of GST substrate(s) 1-chloro-2,4-dinitrobenzene and/or 1,2-epoxy-3-(p-nitrophenoxy)propane, indicating that these 17 GSTs are functional drug-metabolizing enzymes. These results suggest that the 12 GST genes examined in this study are expressed and encoded functional enzymes in cynomolgus macaque.