Aims: For identification of members of the fungal order Eurotiales, an 18S rRNA gene-based oligonucleotide microarray was developed and optimized. Methods and Results: Eurotiales-specific probes covering most members of the Eurotiales as well as species-specific probes were designed and evaluated with three pure cultures (two fungi from the Eurotiales and one fungus from the Hypocreales). Nearly complete 18S rRNA genes of each reference culture were amplified and fluorescently labelled by random priming. Conclusions: Positive and negative hybridization results confirmed that the Eurotiales probes tested in this study could correctly identify members of the Eurotiales. The species-specific probes were also capable of species-level detection. Significance and Impact of the Study: These findings demonstrate the potential applications of a phylogenetic oligonucleotide microarray approach to characterizing fungal species and populations in environmental and other samples.