In this study, we developed a seamless gene deletion method through a two-step integration protocol to construct an industrial baker's yeast with NTH1 deletion. A fusion fragment consisted of the upstream sequence, and the downstream sequence of NTH1 was subcloned into an integrating plasmid containing a URA3 counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the genomic NTH1 locus of recipient baker's yeast, leading to tandem repeats of the upstream flank and the downstream flank. Pop-out of the URA3 marker occurs by integration recombination between either the downstream flank repeats or the upstream flank repeats. Integration recombination between the repeats results in NTH1 deletion without any heterologous DNA and reversion to a wild-type strain. The desired deletion occurred with a frequency of approximately 10(-5). Polymerase chain reaction verification and sequence analysis confirmed the NTH1 disruption and the absence of integrated plasmid sequences in the genome of the selected strain. In addition, the mutant with NTH1 deletion exhibits a higher trehalose accumulation and consequently displays a higher viability of yeast cells after freezing. Thus, this method proposes a protocol to construct mutant yeast without leaving behind any heterologous DNA sequences and will facilitate the genetic engineering of any yeast.