Cladistic analysis is used to study the evolution of 29 biological characters related to nesting behavior, nest provisioning, oviposition, cocoon spinning, defecation, and life cycle (nest characters) in 11 species of Osmia bees (Hymenoptera: Megachilidae) and two outgroup genera. A molecular phylogeny based on 38 allozyme loci-as-characters and a combined character phylogeny are produced to compare with the nest character phylogeny. All phylogenetic trees support the monophyly of the genus Osmia and the subgenus Osmia (Osmia) with Osmia ribifloris Cockerell basal to the other species in this subgenus. The subgenus Helicosmia (=Chalcosmia) is resolved as a sister clade to O. (Osmia) in the molecular tree and as sister to the subgenus Cephalosmia in the nest character and combined trees. Incomplete biological information from additional species of all three subgenera is provided to support the consistency of nest characters at the subgeneric level and their use in the establishment of subgeneric phylogenies in the family Megachilidae.