Legionella pneumophila are gram negative pathogenic water organisms with worldwide significance. Current methods for detection of these pathogens are time and/or cost intensive. We describe a miniaturized detection setup which has the advantage of lower amount of reagents and rapid analysis. A multiplex polymerase chain reaction (multiplex PCR) for direct on-line discrimination of genus and species was established. The identification of different Legionella species is demonstrated, based on real-time fluorescence read out with the intercalating dye EvaGreen. The 16S rRNA region of these water pathogens was used for identification. To show applicability of this detection system, the chip was combined with a temperature controlling unit and a fluorescence intensity measurement. Concentrations of DNA templates up to 2.0 pg/μl were successfully detected and identified. The presented results show that the PCR-chip is suitable for a robust and cost-efficient DNA analysis of Legionella.