Simultaneous saccharification and fermentation (SSF) is capable of performing enzymatic saccharification and fermentation for biofuel production in a single vessel. Thus, SSF has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Here, we describe the application of Pichia kudriavzevii NBRC1279 and NBRC1664 to SSF for bioethanol production. When each strain was incubated for 144 h at 35 °C with Japanese cedar particles, the highest ethanol concentrations were reached 21.9 ± 0.50 g/L and 23.8 ± 3.9 g/L, respectively. In addition, 21.6 ± 0.29 g/L and 21.3 ± 0.21 g/L of bioethanol were produced from Japanese eucalyptus particles when each strain was incubated for 144 h at 30 °C. Although previous methods require pretreatment of the source material, our method does not require pretreatment, which is an advantage for industrial use. To elucidate the different characteristics of the strains, we performed genome sequencing and genome comparison. Based on the results of the eggNOG categories and the resulting Venn diagram, the functional abilities of both strains were similar. However, strain NBRC1279 showed five retrotransposon protein genes in the draft genome sequence, which indicated that the stress tolerance of both strains is slightly different.