Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U & C) and UCN (N = U, C, A, & G), each possessing an unusual secondary structure; tRNASerGCU (for AGY) lacks the entire D arm, while tRNASerUGA (for UCN) has an unusual cloverleaf configuration. We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure. Recombinant mt SerRS clearly footprinted at the TYC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TYC loop sequence in each isoacceptor. However, in the case of tRNASerUGA, TYC loop–D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms. mt SerRS could slightly but significantly misacylate mitochondrial tRNAGln, which has the same TYC loop sequence as tRNASerUGA, implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.