Berkeep is a naturally fermented yoghurt-like milk product consumed as common food in parts of the Darfur State, Western Sudan. Its production and consumption derive much food security and economic benefits to the rural people in the region. The objective of this study was to isolate and characterize yeasts of Berkeep by both conventional and molecular-based methods. A total of 220 yeast isolates, were obtained from 75 samples of Berkeep from three production sites namely, Elkoumah, Manwashy and Kass in Darfur state, were purified then characterized to species level by molecular tools. Based on ITS-PCR using the forward primer Cy5-Y-ITS1 and reverse primer Y-ITS4, the isolates were screened into five groups. Representative of each group was selected for sequence analysis of ITS rRNA gene. The gene sequence was subjected to nucleotide BLAST. The yeast microbiota of Berkeep was dominated by Kluyveromyces marxianus (YG4, 85 isolates) which showed 100% homology towards Kluyveromyces marxianus isolate QKM4. 14.54% (32 isolates), Sixty-eight (Group YG5) identified as Saccharomyces cerevisiae showed 97.22% homology towards Saccharomyces cerevisiae isolate JKSP39. Group YG3 revealed 100% homology towards Pichia kudriavzevii isolate Sugarcane. Eleven isolates, (YG2) showed 99.37% homology towards Pichia kudriavzevii strain YS179. The twenty-four isolates of group, (YG1) recorded 99.70% similarity towards Candida intermedia isolate B-NC-12-f 16i. Very minute variation was observed with in the three production sites concerning the yeast count and type of yeast species.