There are numerous fungal and oomycete pathogens that cause severe damage to solanaceous crops. Rapid and accurate detection and identification of these pathogens is critical for plant disease management. Recently, DNA array technology has been successfully applied for simultaneous detection of multiple microorganisms from various habitats. The goal of this project was to develop a multiplex detection and identification system for major fungal and oomycete pathogens of solanaceous crops. To facilitate this goal, we used a membrane-based macroarray technology that included at least two specific oligonucleotides per pathogen. Based on the internal transcribed spacer sequences of the rRNA genes, 105 oligonucleotides (17 to 27 bases long) specific for 25 pathogens of solanaceous crops were designed and spotted on a nylon membrane. The array was tested against the 25 target pathogen species, 46 infected field samples, and a number of nontarget species. Our results indicate that the oligonucleotide-based macroarray detection system is a reliable and effective method for pathogen detection and identification even when multiple pathogens are present in a field sample.