How animals recognize conspecific individuals has important outcomes in many contexts, but interactions among group members are particularly important. Two recognition criteria are often implicated in these interactions: kin recognition is based on relatedness cues and nestmate recognition is based on familiarity. For social insects, both types of recognition are possible, as many nestmates are close kin and familiarity can develop among individuals that encounter each other repeatedly. To discern whether social insects use kin or nestmate recognition, it is necessary to simultaneously assess how relatedness and familiarity influence behaviour. The facultatively social eastern carpenter bee, Xylocopa virginica, offers an excellent opportunity to study how either nestmate or kin recognition (or both) may influence interactions among nestmates, as many females disperse from their natal nests in spring, and often attempt to join new colonies that may contain unrelated individuals. This leads to frequent behavioural interactions among females that may be related or unrelated, and familiar or unfamiliar. We used observation nests and microsatellite loci to assess the influence of familiarity and relatedness on behavioural interactions during the early phase of colony development, when females establish reproductive queues prior to brood production. Females were more likely to feed and were less aggressive to familiar rather than related nestmates, regardless of their relatedness. This suggests that eastern carpenter bees primarily use learned cues to discriminate among nestmates. Interactions with nestmates were also context-dependent, as females returning to the nest without food were the recipients of more aggression than those returning with food. If spring dispersal leads to reduced relatedness in X. virginica colonies, then nestmate recognition based on familiarity would be an important factor in maintaining group cohesion.