Accurate bacterial identification is essential in various research fields. Traditional methods, culturing bacteria and phenotypic analysis are time-consuming and laborious while the molecular technique using 16S rRNA gene sequence offers a faster and more precise approach. This study investigated the specificity of universal 16S rRNA primers for bacterial identification. Two-step in silico analysis was conducted using Vibrio parahaemolyticus (GenBank accession EU636231) as a model. First, ten universal primer sets were evaluated for their in silico amplification against the target sequence using BioEdit software to assess their ability to specifically bind to the desired region of the 16S rRNA gene. Second, the in silico amplified products from each primer set were then subjected to a BlastN search against the NCBI database to determine the specificity of the amplified sequence. All primers exhibited some degree of specificity during the in silico amplification step. However, significant variations were observed in BlastN results. Notably, primers targeting longer 16S rRNA regions (>500 bp) demonstrated greater concert in achieving species-level resolution. Four primer sets (20F-1500R, 27F-1492R, V1–V3, 8F-534R) perfectly matched the target and showed high identity (>99.5%) within Vibrio. Conversely, other primers displayed lower specificity. V3–V4 and 343F-798R matched multiple Vibrio species, while V4–V5 and 517F-962R even matched other genus beyond Vibrio. These findings demonstrated that primer selection influence the accurate bacterial identification with 16S rRNA gene sequencing. Using primers targeting longer 16S rRNA regions can significantly improve the ability to distinguish between bacterial species at the species level.