Background: Ghee, known as "roghane heiwâni," or "Kermanshahi oil" is a traditional fermented butter-like product highly esteemed for its nutritional value. Ghee is prepared using traditional methods and has substantial potential as a reservoir of probiotic microorganisms. Previous research delved into isolating and identifying lactic acid bacteria (LAB) in Kermanshahi through culture and PCR sequencing. This study seeks to elucidate the microbial profiles and diversity within Kermanshahi using culture, Denaturing Gradient Gel Electrophoresis (DGGE), and sequencing methodologies., Methods: Twenty samples of Kermanshahi oil were meticulously gathered from diverse locales across Kermanshah province. These samples were cultivated under specialized conditions in MRS and M17 environments spanning 24 to 72 h. Following DNA extraction, amplification of the 16SrRNA gene sequences was performed, culminating in sequencing for conclusive identification of the isolates. Furthermore, the DGGE technique was directly employed to separate and identify various species present in the oil samples utilizing bioinformatics software., Results: Sequencing outcomes revealed a diverse array of microorganisms among the isolates, with Lactobacillus constituting 43%, Streptococcus comprising 27.6%, Enterococcus at 4.61%, and yeasts at 7.6%. Other species exhibited lower frequencies, encompassing Rhizobium, Bacillus coagulans, and Staphylococcus hominis., Conclusions: The isolation of a diverse spectrum of probiotic microorganisms underscores their potential utility in the realm of industrial dairy product production. These findings allude to the possibility of integrating these valuable microorganisms, which have historically been associated with traditional products, into the contemporary dairy industry. As consumer interest in probiotic-enriched products surges, the insights gained from this study pave the way for harnessing the benefits of Kermanshahi-derived probiotics., (© 2024. The Author(s).)