Vibrio parahaemolyticus carrying plasmid encoding toxins PirA and PirB is one of the causative agents leading to the severe disease of AHPND in shrimp aquaculture. However, there is a lack of deep understanding of the host-resistant characteristics against V. parahaemolyticus infection. Here, we established a method to obtain hemocytes from shrimp with different V. parahaemolyticus -resistant abilities and performed comparative transcriptome analysis on the expression profiles at the background level of hemocytes from shrimp in two independent populations. Principal component analysis and sample clustering results showed that samples from the same population had a closer relationship than that from shrimp with similar disease-resistant abilities. DEGs analysis revealed that the number of DEGs between two populations was much more than that between V. parahaemolyticus -resistant and susceptible shrimp. A total of 31 DEGs and 5 DEGs were identified from the comparison between V. parahaemolyticus -resistant and susceptible shrimp from populations 1 and 2, respectively. DEGs from population 1 were mainly cytoskeleton-related genes, metabolic related genes, and immune related genes. Although there was no DEGs overlap between two comparisons, DEGs from population 2 also included genes related to cytoskeleton and metabolism. The data suggest that these biological processes play important roles in disease resistance, and they could be focused by comprehensive analysis of multiple omics data. A new strategy for screening key biological processes and genes related to disease resistance was proposed based on the present study.