Abstract The significance of DNA variations among individuals, including single nucleotide polymorphisms (SNPs) and/or genome nucleotide mutations as well as to their detection by using new technology, will improve and facilitate the knowledge of each gene sequence. Microarray may provide information about thousands of gene simultaneously, leading to a more rapid and accurate genotyping. In this view, we developed a new methodology as an example for the detection of SNPs based on DNA microarray, using a panel of HLA alleles representative of loci A, B, DRB1. A panel of 180 oligonucleotide probes was selected to identify polymorphic positions located in exons 2 and 3 of HLA-A and B, and in exon 2 of HLA-DRB1 locus. Each oligonucleotide sequence was designed with a nucleotide mismatch located in the same position as the center of the hybridization sequence. Hybridization experiments were carried out with genomic probes constructed with an asymmetric PCR strategy. The amplified DNAs were obtained from bone marrow cells of donors previously typed for transplant. The results obtained were showing that the method was reliable thus providing a feasible technique both for HLA typing and for the investigation of other regions of genetic and clinical interest including polymorphisms correlated with different autoimmune diseases.