The number of spoilage incidents in the food industry attributable to a species of the genus Moniliella has recently increased, but the risk of food spoilage has not yet been evaluated. The purpose of this study was to develop a method to rapidly identify high-risk species and to conduct a risk analysis study of Moniliella spp. Acetic acid resistance of M. acetoabutens and ethanol resistance of M. suaveolens were higher than for other Moniliella species. All examined strains of M. acetoabutens developed a high tolerance to acetic acid by being cultured twice in liquid media containing low concentrations of acetic acid. These findings indicate that M. acetoabutens and M. suaveolens are high-risk species for food spoilage and must be discriminated from other fungi. We developed species-specific primers to identify M. acetoabutens and M. suaveolens using the polymerase chain reaction (PCR) to amplify the D1/D2 domain of 28S rDNA. The PCR using the primer sets designed for M. acetoabutens (Mac_F1/R1) and M. suaveolens (Msu_F1/R1) was specific to the target species and did not detect other fungi involved in food spoilage or environmental contamination. This method is expected to be effective for the monitoring of raw materials and components of the food production process.