Since 1997, the molecular basis of six different types of Congenital Disorders of Glycosylation with a defect in the synthesis of N-glycans (CDG-I) has been identified. To assure an efficient molecular diagnosis of the six genes involved in these types, we established a denaturing high-pressure liquid chromatography (DHPLC) screening procedure. Primers were designed and conditions were optimised for the analysis of each exon of the PMM2, MPI, ALG6, ALG3, DPM1 and MPDU1 genes. Forty previously described PMM2 mutations were tested to evaluate the method. All of them could be detected. Hence, the sensitivity of the technique is virtually 100%. Screening of 17 novel cases with a tentative, clinical diagnosis of CDG-Ia identified mutations on both alleles in 14 of them, thereby confirming the diagnosis. Six of these mutations were not previously reported (G15E, G42R, Y64C, E93A, G214S and D223N). Sequencing of the complete coding sequence of PMM2 in the remaining three patients did not reveal mutations, corroborating the good performance of the DHPLC method. A similar DHPLC approach was also applied to CDG-Ib, CDG-Ic, CDG-Id, CDG-Ie and CDG-If samples. New mutations were identified in MPI (Y129C) and ALG6 (G227E). All results were confirmed by sequencing. We conclude that the DHPLC platform is a sensitive and efficient method for the rapid analysis of disease genes with a limited number of exons.