We report here the results of mtDNA analysis of remains exhumed in July, 1995 from Mt. Olivet Cemetery in Kearney, Nebraska, that are thought to be those of Jesse James. The remains were poorly preserved, presumably due to wet and slightly acidic soil conditions, and insufficient DNA for analysis was obtained from two bone samples. However, two of four teeth, and two hairs recovered in 1978 from the original burial site on the James Farm, did yield reproducible mtDNA sequences. These mtDNA sequences from the teeth and hairs were all identical, suggesting that they came from the same individual; furthermore, this mtDNA sequence was identical to mtDNA sequences determined from blood samples from two maternal relatives of Jesse James. Therefore, either the remains are indeed those of Jesse James, or they are from an unrelated individual who, by chance, happens to have the same mtDNA sequence. To assess the probability that an unrelated individual would have the same sequence, we searched the forensic mtDNA database, and found that this sequence does not appear among the 2426 mtDNA sequences therein. Hence, the mtDNA analysis supports the identification of the exhumed remains from Mt. Olivet Cemetery as those of Jesse James.