Yeasts were isolated from four potential sources, sweet sorghum juice, sugar cane juice, grapes and rambutan. The 27 yeast isolates were tested for their ethanol tolerance (15% v/v of ethanol) and ethanol fermentation performance in a synthetic ethanol production medium (200 g/L of total sugar). Only five isolates, SCJ04KKU, SCJ07KKU, SCJ09KKU, SCJ14KKU and SSJ01KKU could tolerate 15% ethanol and produce ethanol at levels higher than 55 g/L. The ethanol production efficiency from sweet sorghum juice under high gravity (HG, 200 and 240 g/L of total sugar) and very high gravity (VHG, 280 g/L of total sugar) conditions of the five isolates was tested. Saccharomyces cerevisiae NP01 and S. cerevisiae ATCC4132 were used as reference strains. The results showed that the SSJ01KKU isolate gave the highest ethanol production efficiency under all conditions. Ethanol concentration (PE), yield (YP/S) and productivity (QP) values were 98.89 g/L, 0.50 and 1.18 g/L·h, respectively, with sugar consumption (SC) of 98.96% under the HG condition at 200 g/L of total sugar. Under the HG condition at 240 g/L of total sugar, the PE, YP/S and QP values were 118.12 g/L, 0.51 and 1.41 g/L·h, respectively, with the SC of 95.79%. These values were 82.29 g/L, 0.34 and 0.98 g/L·h, respectively, with the SC of 85.59% under the VHG condition. Addition of urea into the sweet sorghum juice under all conditions significantly shortened the fermentation time, resulting in increased QP values. Based on molecular taxonomic analysis of the five isolates using sequence analysis of the D1/D2 domain and the ITS1 and ITS2 regions, SSJ01KKU is S. cerevisiae, whereas SCJ04KKU, SCJ07KKU, SCJ09KKU and SCJ14KKU are Pichia caribbica.