Paenibacillus mucilaginosus, one of the typical silicate bacteria, has long been used as a biofertilizer in agriculture and has recently shown potential in bioleaching and wastewater engineering. There has been considerable research involving the isolation of P. mucilaginosus for various utilizations; therefore, rapid identification of this species is of great interest. Herein, we describe a specific polymerase chain reaction (PCR) method developed for a rapid identification of P. mucilaginosus, which might provide potential utilization in the investigation of populations, detection of biofertilizers, and identification of novel isolates on a large scale. A gyrB-targeted species-specific primer pair, F2 (5'-ACG GAT ATC TCC CAG ACG TTC AT-3') and R5 (5'-ACG GGC ACG CTG CGC CTG TAC G-3'), was successfully designed to selectively amplify a 519-bp amplicon from P. mucilaginosus. Good specificity was demonstrated by both reference strains and total soil deoxyribonucleic acid, from which only the gyrB gene of P. mucilaginosus was amplified. The detection limit was 4-10 cells per assay. Using the culture-PCR method, 20 of 26 soil isolates on a nitrogen-free medium were rapidly identified as P. mucilaginosus, which was confirmed by sequencing of the gyrB gene.