Classic strain development that combines random mutagenesis and selection has a long history of success in generation of biocatalysts with industrially designed traits. However, the genetic loci contributing to the phenotypic strain changes are difficult to identify prior to genome sequencing technology advancement. In this chapter, we present the approach using Roche 454 next-generation pyro-resequencing to identify the genotypic changes such as single nucleotide polymorphisms (SNP) associated with an ethanol-tolerant strain of Clostridium thermocellum. The parameters used to filter the pyro-resequencing output for SNP identification are also discussed. These can help researchers to identify the genotypic change of other biocatalysts for strain improvement through metabolic engineering.