Although broiler ascites syndrome (AS) has been extensively studied, its pathogenesis remains unclear. The lack of cardiopulmonary function in broilers causes relative hypoxia in the body; hence, the lung is the main target organ of AS. However, the transcriptome of AS lung tissue in broilers has not been studied. In this study, an AS model was successfully constructed, and lung tissues of three AS broilers and three healthy broilers were obtained for RNA sequencing (RNA-seq) and pathological observation. The results showed that 614 genes were up-regulated and 828 genes were down-regulated in the AS group compared with the normal group. Gene Ontology (GO) functional annotation revealed the following up-regulated genes: FABP4, APLN, EIF2AK4, HMOX1, MMP9, THBS1, TLR4, BCL2; and down-regulated genes: APELA, FGF7, WNT5A, CDK6, IL7, IL7R, APLNR. These genes have attracted much attention in cardiovascular diseases such as pulmonary hypertension. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that multiple metabolic processes were enriched, indicating abnormal lung metabolism of AS in broilers. These findings elucidate the potential genes and signal pathways in the lungs of broilers with AS and provide a potential target for studying the pathogenesis and preventing AS.