Dried blood spots are used for newborn screening because of ease of sample collection, handling, and shipment. DNA is stable and accessible in the filter paper matrix. Genotypic confirmation using initial specimens is demonstrated for a regional screening program. Seventy-five blinded samples underwent DNA analysis after Hb electrophoresis. DNA was microextracted from a 1/2-inch semicircle (25 microL whole blood equivalent), amplified, and analyzed by four different methods. Direct amplification without microextraction and automated sequencing from microextracted DNA also was performed. All four analyses agreed for the A and S alleles in 70 of 75 specimens. Three disagreements were clarified by the other semicircle from the original sample: two were due to polymerase chain reaction contamination and one to contamination of one of four analytical tests. Two would have required analysis of a second specimen, one because of polymerase chain reaction failure and the second because the patient had S/beta-thalassemia. Direct amplification without microextraction was successful in an additional 77 of 78 specimens for analysis of the A, S, C, and E alleles. Automated direct sequencing from microextracted DNA was demonstrated for the A, S, and C alleles. Analysis of microextracted DNA from dried blood specimens for A and S alleles reduced the need for and costs of obtaining a second specimen for confirmation by 97%. Direct amplification without microextraction for analysis of A, S, and C alleles permits additional reduction in personnel time and costs. We have demonstrated that microextracted DNA is amenable to automated sequencing after asymmetric polymerase chain reaction. Direct genotypic confirmation can facilitate diagnosis and initiation of medical intervention.