The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.