Sequences from complete mitochondrial cytochrome b genes of 34 tigers support the hypothesis that Sumatran tigers are diagnostically distinct from mainland populations. None of the latter, including Bengals, Siberians, or Indochinese tigers, were found to have fixed diagnostic characters. Phylogenetic analysis of these sequences confirms these results. Within the framework of a phylogenetic species concept, current evidence thus supports the recognition of two distinct taxa, and within the context of this definition they could be ranked at the species level. This paper also documents a previously unrecognized nuclear insert of mitochondrial DNA that includes, minimally, mitochondrial homologues of a control region that lacks the feline mitochondrial repeat sequences, a complete cytochrome b gene, and complete tRNAThr and tRNAsurPro genes. In a phylogenetic analysis of the nuclear cytochrome b-like sequences and various feline mitochondrial sequences, the nuclear insert clusters with lion mitochondrial cytochrome b sequences, which suggests the insert is at least as old as the split between lions and tigers. The results of this study emphasize the importance of doing more to conserve Sumatran tigers. Because they are underrepresented in zoos relative to Bengals and Siberians, an effort should be made to increase captive breeding stocks of Sumatrans. That Sumatrans are a distinct taxonomic entity relative to mainland populations can be used in educational programs to increase conservation efforts within Indonesia.