Aims: This study was aimed to characterise nine clinical isolates in our culture collection that were categorized as Diutina species based on their molecular genetic profiles. D. rugosa is a species complex comprising four taxa., i.e., D. rugosa sensu stricto, D. pseudorugosa, D. neorugosa and D. mesorugosa. The most commonly used phenotypic identification methods for yeasts often lead to the misidentification of this species complex. Methodology and results: The Diutina isolates were received from two local referral hospitals as pure cultures. Species confirmation was performed using conventional phenotypic methods; CHROMagar and RapID Yeast Plus Kit. To study the inter- and intraspecific relationships among the clinical isolates, ITS region, D1/D2 domain and random amplified polymorphic DNA (RAPD) analyses were performed. The results were further validated using the housekeeping gene sequence similarity technique coupled with pairwise sequence alignment. The results from phenotypic methods results were ambiguous and inconclusive. The sequence analyses of ITS regions and D1/D2 domains revealed that the samples consisted of three yeast species; D. rugosa complex: D. rugosa (n=1), D. mesorugosa (n=6), Candida pararugosa (n=1) and Meyerozyma guilliermondii (n=1). The RAPD analysis with random primers, OPG4, OPG11 and OPA18, demonstrated good banding patterns that could distinguish between the Diutina isolates. The pairwise sequence alignment revealed that the Diutina isolates were genetically similar to D. rugosa ATCC 10571. Conclusion, significance and impact of study The molecular methods, D1/D2 domain, ITS1 and ITS4 region, and RAPD analyses have proven helpful for accurately identifying the yeasts, especially closely related species; D. rugosa and D. mesorugosa.