Analysis of the prevalence rates of the endosymbiotic bacterium of the Wolbachia genus in the populations of corn borers of the Ostrinia genus was carried out. Samples of genomic DNA were extracted from field-collected larvae. The PCR analysis for the presence of Wolbachia infection was performed independently using three diagnostic loci: 16SrRNA, gatB, and fbpA. A positive signal for Wolbachia was obtained for 13.5% of the Ostrinia nubilalis larvae (N = 141) and 31.9% of the Ostrinia scapulalis larvae (N = 138). The infection rate ranged from 2.9% (N = 34) to 65.8% (N = 38) in different geographical populations. At the same time, a significantly higher infection rate of O. scapulalis than O. nubilalis was observed in three out of four habitats. It was also found that the Wolbachia infection was more common for O. scapulalis individuals feeding on hemp than for those collected from mugwort.