The Wied's black-tufted-ear marmoset, Callithrix kuhlii, is a New World monkey that lives in tropical and subtropical forests of eastern Brazil. In this study, the mitochondrial genome sequence of this species is determined, which is found to be 16 500 bp in length and consists of 22 transfer RNA genes, 13 protein-coding genes, 2 ribosomal RNA genes and one D-loop control region. The overall nucleotide composition is 32.73% of A, 26.80% of T, 27.04% of C and 13.43% of G, with an AT content of 59.53%. The genome structure about gene order and composition of C. kuhlii mitochondrial genome is similar to those of most other vertebrates. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all the other genes are encoded on the heavy strand. Phylogenetic analysis shows that C. kuhlii and C. jacchus are more closely related to each other than to other marmosets of the genus Callithrix that have its mitochondrial genome published.