Tularemia is an acute infectious disease classified as a natural focal infection, requiring continuous monitoring of both human and animal morbidity, as well as tracking of pathogen circulation in natural reservoirs and vectors. These efforts are essential for a comprehensive prevention and containment strategy. The causative agent, Francisella tularensis, comprises three subspecies-tularensis, holarctica, and mediasiatica-which differ in their geographic distribution and virulence. The ability to directly detect the pathogen and differentiate between subspecies has enhanced diagnostics and allowed a more accurate identification of circulation areas. Real-time PCR protocols for identification of F. tularensis subspecies tularensis and holarctica have been developed, utilizing specific primers and probes that target unique genomic regions. In this study, we present the development of a new real-time PCR assay for the detection of Francisella spp. and differentiation of F. tularensis subsp. mediasiatica. The specificity of the assay was tested on DNA from 86 bacterial species across 31 families unrelated to Francisella spp., as well as on DNA collections of F. tularensis subsp. mediasiatica and F. tularensis subsp. holarctica. The limit of detection (LOD95%) for real-time PCR in detecting Francisella spp. was 0.297 fg (0.145 genomic equivalents, GE) for holarctica DNA and 0.733 fg (0.358 GE) for mediasiatica DNA. The LOD95% for subspecies differential identification of mediasiatica was 8.156 fg (3.979, GE). The high sensitivity and specificity of these developed protocols enable direct detection of pathogens in biological and environmental samples, thereby improving the efficiency of tularemia surveillance in Kazakhstan.