The identification of mutations in the MEN1 gene causing MEN1 has represented a challenge since the cloning of the gene in 1997 because of the lack of mutation hot-spots in the gene and the lack of phenotype-genotype correlations. The use of denaturing high performance liquid chromatography (DHPLC), a high throughput, reliable and automated heteroduplex-based technique, is the ideal for mutation detection in MEN1. In this work, DHPLC was optimised for the screening of the nine coding exons and splice junctions of MEN1. Thanks to collaboration between two French laboratories recognised as reference centres for genotypic MEN1 diagnosis (Lyon and Lille), a blind retrospective study conducted in a cohort of 160 unrelated MEN1 probands with (or without) known germline mutations was undertaken to evaluate the sensitivity of DHPLC. We were able to detect 101 different sequence variations by DHPLC, distributed in the 10 analysed DNA fragments and corresponding to 100% of mutation detection compared with direct sequencing. 1.2% of samples were considered as false positive, exhibiting a heterogenous profile. DHPLC did not detect five cases of deletion or duplication of complete exons, neither did direct sequencing, showing the limits of the technique. Nevertheless, the method appeared to allow automated, rapid and low-cost mutation detection with high accuracy. Direct sequencing can be then applied to identify the sequence variations on the targeted DNA fragments showing heterozygous profile by DHPLC. In conclusion, genotypic diagnosis of MEN1 can benefit from DHPLC in terms of efficacy, rapidity and cost.