The diagnosis of mycobacterial co-infection is one of the pressing public health issues. The study was aimed to determine discriminatory power of multiplex PCR used for species identification when detecting mixed mycobacterial populations. The study involved model samples representing the mixtures of DNA of two mycobacterial species with the ratios of 1 : 1, 1 : 9, 1 : 99, and 1 : 999 and different total DNA concentrations (10³ gEq/mL to 10⁶ gEq/mL). The model samples were assessed using the multiplex PCR-based AmpliTube-RV-Differentiation kit (Syntol LLC; Russia). It has been shown that the kit is capable of detecting the mixtures of mycobacterial species with high discriminatory power. The discriminatory power of real-time PCR used for analysis of the mixture of DNA of two mycobacterial species depended on the total DNA content in the sample and varied between 0.1% for high-rate samples (total DNA concentration 10⁶ gEq/mL) and 50% for low-rate samples (total DNA concentration 10³ gEq/mL) and corresponded to the amount of DNA of the species in the sample of at least 5 × 10² gEq/mL. When the amount of DNA of each species in the mixture was at least 5 × 10² gEq/mL, the results of PCR test for detection of co-infection did not depend on the mucobacterial species contained in the mixture, which should be taken into account when analyzing PCR results.