Illumina Genome Analyzer (GA), as one of the new-generation sequencing instruments, can produce large amount (typically 3G per paired-end run) of short reads (30-50bp in length) in several days. The high-throughput is suitable for genome resequencing, which requires deep coverage to detect genomic polymorphisms such as single nucleotide polymorphisms (SNP’s), insertion/deletion events (indels) and structural variations (SV’s). However, the new technology also challenges bioinformatics tools due to intensive computing resource requirements. Traditional alignment and mutation detection pipelines are mainly designed for capillary sequencing, which are not appropriate for new-generation sequencing technology as they may consume unacceptable memory or disk space and cost too long time to finish. Therefore, we have developed a new protocol, which takes full use of characteristics of Illumina GA reads, for genome resequencing and mutation detection. The new protocol runs over ten times faster than traditional method and achieves a high accuracy on detecting polymorphisms.