Objective To develop an oligonucleotide array to detect single nucleotide mutations in 23S rRNA gene.Methods Primers and probes targeting A2142G.A2143G and C2182T mutations in 23S rRNA gene were designed tp develop an oligonucleotide array.Samples were performed by an asymmetric PCR and the PCR products were hybridized with the specific DNA microarray chips.Non fluorescence-labeled PCR products were cloned into T vectors.The results of oligonucleofide array were confirmed by direct DNA sequencing and evaluated by minimal inhibitory concentration (MIC).Results The results obtained from oligonucleotide microarray were identical to those of direct sequencing.In 54 Helicobacter pylori samples,oligonucleotide microarray indicated that no A-to-C transition at 2142 was found,and the mutant rate of A2143G was 11.11 % (6/54),the mutant rate of C2182T was 12.96% (7/54).A2143C,A2143T,C2182A and C2182G mutations were not found.The other specimens were wild-type.All the above results were the same as that of MIC tests.Conclusions The oligonucleofide microarray is a reliable and accurate genotyping assay for clarithromycin-resistance of Helieobaeter pylofi.It is high-throughput screening method for gastric mucosa and improve the application of strategy for personalized therapy.