Yeast strains present an attractive potential for the production of organic acids. Citric acid is a commercially valuable organic acid, widely used in food, pharmaceutical and beverage industries. In this study, 260 yeast isolates were successfully isolated from the south region of the Kingdom of Saudi Arabia. All yeast isolates were primary screened for their ability to produce organic acids using bromocresol green agar plates test. A total of 251 yeast isolates were showed positive results with yellow halo production around the colonies. Further screening was performed to evaluate the citric acid production by twenty promising isolates using free and immobilized cells techniques. Results showed that, the productivity of citric acid by immobilized cells (30- 40 g/L) was greater than that of the freely suspended cells (8-19 g/L). The cells immobilized technique with isolate KKU-L42 produced a higher production rate of 62.5% compared with freely cells, while immobilized cells of isolate KKU-L53 was produced about 52.2% increase in citric acid production. Out of 20 isolates, 10 yeasts (KKU-L42, KKU-L53, KKU-L86, KKU-L165, KKU-L190, KKU-L195, KKU-L207, KKUL209, KKU-L224 and KKU-L258) were selected based on their production of citric acid by the two methods. The exact identification of the 10 selected isolates was detected on the basis of D1/D2 domain of the 26S rRNA gene amplification and sequence determination. Alignment results and the comparison of 26S rRNA gene sequences of the isolates to 26S rRNA gene sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate position of the isolates as Candida tropicalis KKU-L42, Pichia kluyveri KKU-L53, Pichia..