Mutations in the α-galactosidase A (α-Gal A, GLA) gene cause Fabry disease, an X-linked recessive lysosomal storage disease. The majority of mutations are private, and confirmation of carrier status in females requires the definitive identification of a DNA mutation. In addition, knowledge of a family's mutation enables rapid and precise preimplantation and prenatal genetic testing. Here we report the development and use of DHPLC to rapidly and cost-effectively screen for α-Gal A mutations. Optimal DHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to the seven α-Gal A exons and their adjacent intronic/flanking sequences. At least five known mutations in each exon (45 in total) were screened by DHPLC to validate the method. Mutation detection was then performed in 14 affected males diagnosed by enzyme assay and 39 at-risk females, and the amplicons with abnormal DHPLC profiles were sequenced. In all affected males, and in 32 of the 39 at-risk females, four and 16 previously reported and 10 and 15 new mutations were identified, respectively. Sequencing all seven α-Gal A gene amplicons in the seven at-risk females who had normal DHPLC profiles excluded them as mutation carriers. Only one mutation (p.P362L) was not initially identified by its DHPLC profile, but in retrospect the profile was abnormal, emphasizing the need for experience in inspecting the profiles. In addition, this technique detected two new intronic polymorphisms, c.640–16A>G and c.1000–22C>T, with frequencies of 0.14 and 0.25 in both normal individuals and Fabry patients, respectively. This DHPLC method should improve the rapidity and cost-effectiveness of α-Gal A mutation identification in affected males and carrier females for Fabry disease. Hum Mutat 25:299–305, 2005. © 2005 Wiley-Liss, Inc.