White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally, and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the South-East (Cox’s Bazar) and South-West (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox’s Bazar and Satkhira), and over the study periods (2017-2019). The mean Log load of WSSV varied from 8.40 (Cox’s Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71±0.76 ppt, 3.7±0.1 ppm, 34.11±0.38˚C and 8.23±0.38, respectively in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at 167th position in the isolates from Cox’s Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 X 109 copies of WSSV per mL resulted a median LT50 value of 73 hrs and 75 hrs, respectively. The in-vivo trial showed higher mean Log WSSV copies (6.47±2.07 per mg tissue) in BD1 challenged shrimp PL compared to BD2 (4.75±0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 hrs and 62 hrs of challenge, respectively with mean Log WSSV copies of 12.06±0.48 and 9.95±0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs) penaeidin and lysozyme expression was lower in BD1 challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.