The random bacterial genomic DNA microarray chip (RBGDMC), which was fabricated using random genomic DNA fragments obtained from the fragmentation of bacterial genome by using four different pairs of restriction enzymes, was found to discriminate bacterial species in the same genus and resulted in the determination of dominant bacteria in enriched cultures. The identification of a dominant bacterial species was successfully conducted in the co-culture of three different bacteria using the RBGDMC. In addition, the analysis of the chip data could confirm if any of the selected bacteria is the most abundant or if some bacteria were enriched and became the dominant species within the consortium after the samples were prepared from the repeated cultures of real sludge in a complex medium. This study shows the successful implementation of the RBGDMC for the identification and monitoring of dominant bacteria in complex environmental bacterial communities simply without any PCR amplification of the target nucleic acids.