The most conventional approach for evaluating genetic variability in an insect population involves assessing the degree of enzyme polymorphism. Hymenoptera display a relatively low level of genetic variability compared with most insect species. DNA probes consisting of tandemly repeated sequences are powerful tools for detecting polymorphisms when employed to develop DNA fingerprinting (DNAfp) profiles in a wide range of organisms. This report describes genetic variability in the solitary bee species Megachile rotundata as assessed by DNAfp using the Ml3 sequence and a synthetic oligonucleotide sequence homologous to a hypervariable region of the α-globin gene. DNAfp comparisons among offspring were used to analyze genealogical structure in M. rotundata nests. The results indicate that polyandry, by a large number of males, is not a common phenomenon in M. rotundata bee species. In the present analysis, it is likely that the broods raised in single nests are mostly the offspring of one singly mated female. However, the data does not preclude that for certain nests two males could have been involved in the mating process.Key words: Megachile rotundata, DNA fingerprinting, M13 sequence, α-globin hypervariable sequence, intra-nest genetic relationships.