Abstract: To systematically investigate the characteristics of white spot syndrome virus (WSSV)-derived small interfering RNAs (vsiRNAs) in different crustacean hosts, publicly available small RNA and miRNA sequencing datasets of crustaceans were retrieved and analyzed from the NCBI SRA database. After data preprocessing, all reads were stringently mapped to the WSSV reference genome using a zero-mismatch strategy. Based on the mapping results, five representative crustacean hosts, Penaeus vannamei, P. penicillatus, Scylla paramamosain, Carcinus maenas, and Procambarus clarkii, were selected for analysis of WSSV-derived vsiRNA characteristics. The results showed that abundant WSSV-derived vsiRNAs were detected in P. vannamei and C. maenas, with a predominant length of 22 nt and distribution on both the positive and negative strands of the viral genome, exhibiting typical features of small interfering RNAs. In contrast, the other three hosts displayed much lower amounts of WSSV-derived vsiRNAs with dispersed length distributions and no obvious dominant peak. Although overall distribution patterns of WSSV-derived vsiRNAs varied among hosts, shared genomic regions of WSSV consistently exhibited vsiRNA enrichment across multiple species. Sequence and structural features of these hotspot regions, including elevated GC content, lower minimum free energy, and enrichment of conserved motifs forming double-stranded or hairpin structures, suggest that RNA secondary structure may contribute to the preferential processing of these regions by the RNAi pathway. This study provides a comparative overview of WSSV-derived vsiRNA characteristics in five crustacean species based on public sequencing data, and offers insights into crustacean RNAi responses to WSSV infection and a reference for future studies on antiviral mechanisms and RNAi-based disease control strategies.