We assessed enzyme variability in a solitary leafcutter bee, Megachile rotundata (Fabricius), and found sufficient variability to compare relatedness among progeny in a nest in two samples: (1) nests with an associated adult female (putative mother) and (2) nests with out-of-sequence male progeny (M. rotundata, like most cavity-nesting solitary aculeates, usually provisions all female cells in a nest before any male cells). Estimates of relatedness from nests with associated adult females were consistent with the progeny in a nest being closely related and the offspring of a single, once-mated female. The relatedness estimates from nests with out-of-sequence males were all much lower, suggesting some combination of nest parasitism, nest usurpation, nest abandonment, and multiply mated females in these nests. The high levels of relatedness among female progeny in the first sample, as expected for full sisters in a haplodiploid system, are considered with respect to the evolution of social behaviour in the Hymenoptera. Besides higher estimates of relatedness, the nests with an associated female also had a lower male:female sex ratio than did nests with out-of-sequence males.