The limited information on differential gene expression in the different serotypes of Actinobacillus pleuropneumoniae has significantly hampered the research on the pathogenic mechanisms of this organism and the development of multivalent vaccines against A. pleuropneumoniae infection. To compare the gene expressions in the A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3), we screened the differentially expressed genes in the 2 strains by performing representational difference analysis (RDA). Northern blot analyses were used to confirm the results of RDA. We identified 22 differentially expressed genes in the CVCC259 strain and 20 differentially expressed genes in the CVCC261 strain, and these genes were classified into 11 groups: (1) genes encoding APX toxins; (2) genes encoding transferrin-binding protein; (3) genes involved in lipopolysaccharide (LPS) biosynthesis; (4) genes encoding autotransporter adhesin; (5) genes involved in metabolism; (6) genes involved in the ATP-binding cassette (ABC) transporter system; (7) genes encoding molecular chaperones; (8) genes involved in bacterial transcription and nucleic acid metabolism; (9) a gene encoding protease; (10) genes encoding lipoprotein/membrane protein; and (11) genes encoding various hypothetical proteins. This is the first report on the systematic application of RDA for the analysis of differential gene expression in A. pleuropneumoniae serotypes 1 and 3. The determination of these differentially expressed genes will serve as an indicator for future research on the pathogenic mechanisms of A. pleuropneumoniae and the development of a multivalent vaccine against A. pleuropneumoniae infection.