Otters are classified in the Lutrinae and are united by a suite of semi-aquatic adaptations that distinguish them from the rest of the Mustelidae. Past systematic studies have been based primarily on overall similarity of morphological characters but have not been concordant, with regard either to relationships within the subfamily or to the relationships of the Lutrinae to other taxa in the Mustelidae. We evaluate the relationships among nine of the 13 species of otters and their position in the Mustelidae through phylogenetic analysis of the complete nucleotide sequence of the mitochondrial cytochrome b gene. Based on the sequence data presented here, our results suggest that otters are divided into three primary clades that include: (1) the North American river, neotropical and marine otters; (2) the sea, Eurasian, spotted-necked, cape clawless and small-clawed otters; and (3) the giant otter. Furthermore, our results indicate that among sampled mustelid taxa, members of the genus Mustela are most closely related to otters. The hierarchical relationships among clades are not well resolved, particularly the monophyly of otters, largely because a pattern of short internal branches combined with long terminal branches suggests a rapid evolutionary radiation. Estimates of divergence time calibrated by the fossil record suggest that the lineages leading to the North American river, neotropical and marine otters, and the giant otter diverged before the end of the Miocene, much earlier than predicted from the fossil record.