Objectives: The present study aimed to develop strategies for genotyping DO*HY (Dombrock system) and DI*A/DI*B (Diego system) alleles and to evaluate the impact of genomic and self-declared ancestry on rare donor screening in admixed populations.Background: The antigens Hy and Di b demonstrate clinical importance. The lack of antisera for the serological evaluation of these antigens makes it necessary to develop molecular methods. In addition, considering that some rare red blood cell phenotypes present differences in frequency between ethnic groups, it is important to assess the applicability of self-declared ancestry in the search for rare donors in admixed populations.Methods: DO*HY and DI*A/DI*B genotyping based on real-time polymerase chain reaction (PCR) was standardised. A total of 457 blood donors clustered by selfdefined skin colour/race categories were genotyped. Furthermore, individual genomic ancestry was used in the analyses. Results:The assays developed are reproducible and provide satisfactory results even at low concentrations of DNA, which make them useful in situations where the DNA is scarce, such as dried blood spots on filter paper, or when screening for pooled samples. No significant difference was observed in the frequencies of the DI*A, DI*B and DO*HY, comparing the self-declared White (branco) donors with those who are Black (preto) and Brown (pardo). Conclusion:Real-time PCR, especially using pooled samples, is a promising strategy to screen rare blood donors. Although both self-reported race/colour and some blood group phenotypes are associated with ancestry, the results point to a greater complexity in the application of self-declared race/colour in the screening of rare donors in admixed populations. K E Y W O R D S ancestry, Diego, Dombrock, rare blood donor Amanda A. Muniz and Adão R. da Silva contributed equally to this study.