The phylogenetic relationships of the Glossiphoniidae (Rhynchobdellida) were investigated using morphological characters and the mitochondrial genes cytochrome c oxidase subunit I and nicotinamide adenine dinucleotide dehydrogenase subunit 1. Thirty-five taxa representing 10 of the 23 currently recognized glossiphoniid genera were sampled, including more than 70% of known North American species, as well as others from Europe, South America, Africa, and a species endemic to Lake Baikal. Outgroup taxa included species from the Piscicolidae and Ozobranchidae. Cladistic analysis resulted in 1 most-parsimonious tree. Subfamily distinctions, i.e., Haementeriinae, Theromyzinae, and Glossiphoniinae, that have been based on eye morphology and reproductive biology are not corroborated. Results also provide insights into several problematic genus-level classifications. For example, relationships of Placobdella and Haementeria are clarified and elimination of Desserobdella may be necessary. Bloodfeeding from vertebrates is seen to be a primitive characteristic that has been lost twice within the clade. The hypothesis that the biannulate leech, Oligobdella biannulata, represents an important transitional form is re-evaluated in a phylogenetic context.