The study aimed to investigate transcriptomic mechanisms underlying natural resistance to avian leukosis virus subgroup J (ALV-J) in chickens by comparing resistant and susceptible individuals using high-throughput RNA sequencing. The PRJNA685043 RNA-Seq dataset available on the FAANG data portal was used for analyses. The gene expression profiles of chicken liver tissues from ALV-J-resistant and ALV-J-susceptible birds (N = 6; 3 resistant and 3 susceptible) were analyzed to identify differentially expressed genes (DEGs), differential transcript usage (DTU), and associated molecular pathways that may contribute to viral resistance. Transcript quantification was performed using kallisto, followed by differential gene expression (DGE) analysis with DESeq2 and DTU evaluation using a combined DRIMSeq–DEXSeq–stageR workflow. Functional characterization and interaction profiling were conducted using STRING and additional regulatory analyses. The analysis identified 36 significant DEGs, including 32 upregulated and 4 downregulated genes in resistant chickens. Key genes were associated with mitochondrial regulation, calcium signaling, DNA damage response, and suppression of tumorigenesis. DTU analysis revealed 22 genes exhibiting significant isoform switching, suggesting alternative splicing as an additional regulatory layer. Interaction network assessment highlighted limited but meaningful relationships among DEGs and DTU genes, indicating that multiple independent pathways may contribute collectively to resistance.