To understand the prevalence of white spot syndrome virus (WSSV) of cultured Penaeus vannamei (P. vannamei) in Guangxi Province and to evaluate the genetic diversity of deletion region ORF23/24 and the genetic evolution lineage among WSSV isolates of Guangxi Province and other places, we collected 306 diseased P. vannamei samples between May 2010 and September 2013 from Beihai, Qinzhou and Fangchenggang City in Guangxi Province, and the detection of WSSV among these samples was carried out using the nested polymerase chain reaction (nested PCR) recommended by Office International Des Epizooties (OIE). We selected 53 positive samples found by first step PCR for amplification of the deletion regions ORF23/24 of the WSSV genome, and 12 PCR amplifications were used to clone and sequence. We then conducted a pairwise and multiple alignment analysis to evaluate the degree of genetic divergence among different strains. The results were as follows: 82 of 306 samples were positive for WSSV, and the positive rate was 26.8%; 12 Guangxi strains were 2096 bp in length and carried a 10970 bp deletion in the ORF23/24 region relative to WSSV-TW, which has the longest nucleotide sequence in this region. By comparison of nucleotide sequences of ORF23/24 region, we found that 12 Guangxi strains were above 99.6% homology, only existed nucleotide substitution but no deletion and insert, and BH-3, QZ-1, FCG-1 and FCG-2 strains were complete homology. In the phylogenetic tree of WSSV deletion region ORF23/24, the Guangxi strains were near in genetics and formed a separate branch with India strain IN-05-Ⅰ, while far from WSSV-TW, WSSV-CN and WSSV-Korea. In conclusion, there was some extent of prevalence of WSSV infection in cultured P. vannamei of Guangxi; the results showed that the Guangxi WSSV strains had large deletion in middle region of the ORF23/24 gene, and there were no differences in spatio-temporal and they were closer in genetics to India strain IN-05-Ⅰ than other strains in GenBank.